Therapeutic curcumin derivatives

ABSTRACT

Curcumin analogs and methods are provided for treatment of disease.

This application claims the benefit of U.S. Provisional Application Ser.Nos. 60/695,046, filed Jun. 29, 2005; 60/787,695, filed Mar. 30, 2006;and 60/787,694, filed Mar. 30, 2006; and further, this application is acontinuation-in-part of U.S. application Ser. No. 11/373,444, filed Mar.10, 2006 which, in turn, is a continuation in part of U.S. applicationSer. No. 11/057,736, filed Feb. 14, 2005 which, in turn, claims thebenefit of U.S. Provisional Application Ser. No. 60/544,424, filed Feb.12, 2004; all of which are incorporated herein by reference in theirentireties.

BACKGROUND

The transcription factor NF-κB is an established regulator of numerousgenes important in the inflammatory response. More recently, activationof NF-κB has been shown to have a role in many aspects of oncogenesisincluding control of apoptosis as well as regulation of cell cycling andcell migration (Yamamoto et al., J. Clin. Invest. 2001, 107, 135;Baldwin, A. S. J. Clin. Invest. 2001, 107, 241). Activated NF-κB hasbeen observed in many cancers and is especially important in metastasis(Andela et al., Clin. Orthop. Relat. Res. 2003, 415 (suppl), S75).

NF-κB is a collective name for dimeric transcription factors comprisingmembers of the Rel family of DNA-binding proteins that recognize acommon sequence motif. NF-κB is also commonly referred to as, forexample, NFκB and NFκB, with the abbreviations being usedinterchangeable. Five members of the mammalian Rel family are known:RelA (p65), RelB, c-Rel, NF-κB1 (p50) and NF-κB2 (p52) (Baldwin, A. S.,Annu. Rev. Immunol. 14, 649 (1996)). The five members to the NFκB familyare distinguished by the presence of a Rel homology domain. Each NFκBmember is retained in the cytosol as a complex, the most prevalent ofwhich is a dimer consisting of the two subunits, p65 and p50. Any homo-and heterodimer is considered NF-κB, although the most commonly found inactivated cells, RelA/NF-κB (p65/p50) heterodimer, is often referred toas “classic” NF-κB. All Rel proteins contain a Rel homology domain (RHD)that is responsible for dimer formation, nuclear translocation,sequence-specific DNA recognition, and interaction with I-κB proteins.RelA, RelB and c-Rel also contain transactivation domains required forthe recruitment of transcriptional machinery, and thus representtranscriptionally active components of NF-κB. FIG. 1A provides apictorial representation of the NF-κB activation cascade.

NF-κB activation is controlled by an interaction with a family ofinhibitors proteins known as I-κB. The I-κB family includes I-κBα,I-κBβ, I-κBγ, I-κBε, p100, p102, and Bcl-3 (Whiteside et al., Semin.Cancer Biol. 8, 75 (1997)). All I-κBs share three common structuralfeatures: an N-terminal regulatory domain, which is responsible for asignal-dependent I-κB proteolysis, a core domain composed of six orseven ankyrin (ANK) repeats mediating an interaction with Rel proteins,and a C-terminal domain containing a PEST motif implicated in basal I-κBturnover.

In unstimulated cells, NF-κB resides in the cytoplasm as an inactiveNF-κB-I-κB complex. I-κB binding hinders recognition of the NF-κBnuclear localization signal by nuclear import machinery, thus retainingNF-κB in the cytoplasm. Stimulation of cells releases active NF-κB,which is now free to enter the nucleus and activate transcription.Release of NF-κB is generally mediated by the degradation of I-κB.

Phosphorylation of IκB by IκB kinase (IKK) in response to an array ofsignals leads to degradation of IκB and the release of NFκB. Free NFκBis translocated to the nucleus where it binds to promoter regions of DNAresulting in the activation of a battery of genes, includingpro-inflammatory genes (cytokines IL1 and TNFα; chemokines; stressresponse genes; and pro-inflammatory enzymes including iNOS, COX-2 andMMP-9). Compounds inhibiting the activation of NFκB can be directed atIKK or at NFκB. IKK inhibitors will prevent phosphorylation of IκBwhereas direct inhibitors of NFκB may block NFκB-DNA interactions. Karinet al., (2004) Nat Rev Drug Discov 3, 17-26. The inducible degradationof I-κB occurs through consecutive steps of phosphorylation,ubiquination, and proteosomal degradation. I-κB processing is controlledby three large multi-protein complexes: IKK or signalsome, I-κBubiquitin ligase, and 26S proteosome (Makarov, S. S., Mol Med Today. 6,441-8 (2000)). Whereas the I-κB ubiquin ligase and the 26S proteosomeare constitutively active, IKK activity is induced upon stimulation.Various stimuli, including inflammatory cytokines, mitogens, viralproteins, and stress, can activate IKK, thereby inducing phosphorylationof two critical serine residues of I-κB. The phosphorylation of I-κBtargets it for rapid ubiquination and proteosomal degradation.

There are two IKK's, designated IKKα and IKKβ, that exist in a complexcalled the IKK signalsome. Also included in the complex are theIKK-associated protein (IKAP) and NEMO (also called IKKγ). There aremany upstream regulators of the IKK signalsome that have been identifiedand could be useful “targets” for suppression of IKK expression and,ultimately, NFκB expression. Thus, compounds that prevent thephosphorylation of IκB (and therefore prevent the activation of NFκB)may act directly on one or more members of the IKK signalsome or mayinhibit upstream kinases, such as SFK or any other such family ofkinases. This complicates structure-based design of potential drugs toprevent activation of NFκB, especially because crystal structures ofmembers of the IKK signal some are not available. It is noteworthy thatthere are also IKK-independent pathways for activation of NFκB.

Most available evidence suggests that IKKβ is the canonical pathway forNFκB activation and that IKKα functions in special circumstances. Karinet al., (2004) Nat Rev Drug Discov 3, 17-26. Whereas binding to IκBβeffectively sequesters NFκB in the cytoplasm, binding to IκBα does notpreclude nuclear translocation. In fact, the NFκB-IκBα trimericcomplexes shuttle between the cytoplasm and the nucleus. The source ofthis difference is that binding of IκBβ to a p50/p65 complex blocks NLSlocated on both NFκB subunits, whereas binding to IκBα blocks only thep65 NLS. Thus, NFκB-IκBα complexes contain both an exposed functionalNLS and several nuclear export signals (NES) found in the N-terminaldomain of IκBβ and in the activation domain of p65. The functions ofboth NLS and NES result in this shuttling between the cytoplasm and thenucleus. However, multiple NES seem to dominate, resulting in aprimarily cytoplasmic localization of NFκB-IκBα complexes. When nuclearexport is blocked with leptomycin B (LMB), the complex accumulates inthe nucleus. Since IκBα is the most prevalent IκB isoform, in mostresting cells the majority of NFκB protein is located in the cytoplasmbound to IκBα. Inflammatory stimuli, such as IL-1 treatment, leads toactivation of IKK activity, phosphorylation of IκBα on serine 32 and 36,recognition of IκBα by the E3 ubiquitin ligase, IκBα ubiquination,degradation of IκBα by the 26S proteasome, and release of NFκB. The twoexposed NLS on NFκB subunits then cause nuclear translocation of thetranscription complex. However, numerous studies have now documentedstates where NFκB activation occurs in the absence of IκBα degradation.

For many stimuli, including interleukin-1 (IL-1), and tumor necrosisfactor α (TNF-α), I-κB degradation and NF-κB nuclear translocation arenecessary, but not sufficient, for the induction of NF-κB dependenttranscription. The ability of NF-κB to initiate transcription depends oninteractions with different transcriptional co-activators (Schmitz etal., J. Biol. Chem. 270, 7219-7226 (1995). Although regulatedindependently, the pathways controlling I-κB degradation and NF-κBtranscription function act in synergy with the activation of NF-κBmediated transcription.

NF-κB appears to play a pivotal role in both initiation and perpetuationof chronic inflammation. CD4⁺ T cells are a trigger of immuneinflammation, and NF-κB appears to be an important mediator ofantigen-induced T-cell activation. Secreted products of activated Tcells and direct cell-cell contacts cause activation of macrophages,fibroblasts, and endothelial cells. Once established,autocrine/paracrine loops of inflammatory cytokines and growth factorsare capable of maintaining the activation of non-immune cells within thelesion, thereby perpetuating the chronic inflammatory process.Persistent NF-κB activation has been found in many chronic inflammatorydiseases, including rheumatoid arthritis, asthma, inflammatory boweldisease, ulcerative colitis, and atherosclerosis (Barnes et al., NewEngl. J. Med. 336, 1066-1071 (1997).

Additionally, the evidence that links activation of NF-κB to oncogenesisis compelling. NF-κB is activated by a number of viral transformingproteins (Hiscott et al., J. Clin. Invest. 2001, 107, 143), andinhibition of NF-κB activation through expression of a dominant negativeIKK can block cell transformation (Arsura et al., Mol. Cell Biol. 200020, 5381). NF-κB activation protects cells from apoptosis induced bycancer chemo-therapeutics and oncogenes (Barkett et al., Oncogene 1999,18, 6910), and activation of NF-κB promotes expression of metastaticfactors (Baldwin, A. S. J. Clin. Invest. 2001, 107, 241). NFκBactivation results in up-regulation of cyclin D1, a cell cycle regulatorthat is up-regulated in many tumors. NFκB is constitutively expressed inmany cancer cell lines. Additionally, a number of dietarychemopreventive compounds such as flavonoids, curcumin, and reserveratolblock activation of NFκB. Further, the expression of interleukin 8(IL-8) which has been identified as a key factor in both angiogenesisand metastasis is very dependent on NFκB activity.

NF-κB is active in many tumors, and expression of NF-κB-responsive genesprovide cancer cells with distinct survival advantages that inhibitcancer treatment. NF-κB is constitutively activated in many cancercells, and NF-κB may also be conditionally activated in both cancercells and stromal cells by the tumor microenvironment. Normally, NF-κBactivation is prevented by binding to inhibitor (IκB) proteins, the mostprevalent being inhibitor of NF-κB alpha (I-κBα). In response toinflammatory cytokines, the release of NF-κB is triggered byphosphorylation of I-κBα on serines 32 and 36, resulting in ubiquinationand degradation of I-κBα protein. However, in cancer cells subjected toenvironmental conditions such as hypoxia, nutrient starvation, orX-rays, NF-κB activation is caused by phosphorylation of I-κBα on atyrosine residue (Tyr42) by Src family kinases (SFKs). Thus, NF-κBactivation via IκBα Tyr42 phosphorylation is expected to occur in solidtumors due to constitutive activation of SFKs such as the Src oncogenein response to the hypoxic and nutrient poor nature of the tumormicroenvironment, or due to radiation treatment of the tumor.

NFκB was first identified as the nuclear factor in mature B-lymphocytesthat binds to an 11 bp element (GGGACTTTCC) within the κ-light chaingene enhancer, but it was soon realized that NFκB is not aB-cell-specific transcription factor. A wide variety of environmentalstimuli and stresses lead to the formation of active NFκB complexeswithin almost every cell type, and NFκB activation mediates thetranscription of over 180 target genes.

There are several NFκB crystal structures for use in structure-baseddrug design including a human NFκB-DNA structure. However, compoundsthat have been reported to inhibit activation of NFκB have generallybeen suggested to work at the level of IKK, rather than to interferewith NFκB-DNA interactions or with NFκB dimerization to prevent itsinteraction with DNA. Given the mechanisms of suppression and expressionof NFκB, compounds inhibiting the activation of NFκB can be directed atIKK, SFK, or other kinases at NFκB-DNA interactions. Kinase inhibitorswill prevent phosphorylation of IκB where direct inhibitors of NFκB mayblock NFκB-DNA interactions. For example, it has been shown recentlythat a new class of retinoid-related drug candidates inhibits IKKdirectly. Bavon et al., (2003) Mol Cell Biol 23, 1061-1074. Bycomparison, a synthetic derivative of the fungal metabolite jesterone,which blocks activation of NFκB, was shown to inhibit a kinase involvedin phosphorylation and activation of IKK (β-subunit). Liang et al.,(2003) Mol Pharmacol 64, 123-131. It appears, therefore, that inhibitionof one or more of the kinases associated with the IKK signalsome may bea promising route to the development of new therapeutic agents that workthrough blocking the activation of NFκB. Further, because NFκBresponsive genes can promote angiogenesis, cell motility and invasion,and block apoptotic cell death, this mechanism represents a considerableobstacle to cancer treatment. Therefore, there is a greatly felt needfor development of small molecule inhibitors of NFκB expression.Particularly, but not exclusively, inhibitors of IκBα Tyr42phosphorylation have vast potential to serve as adjuvant cancertherapeutics.

Activator Protein-1 (AP-1) is another protein transcription factor foundin mammalian cells. AP-1 like NF-κB is a prosurvival andpro-inflammatory protein. AP-1 is an established regulator of numerousgenes important in a variety of cellular processes including cell growthregulation, differentiation and proliferation (Angel et al., Cell 1987,49, 729-739). Growth factors, hormones, tumor promoters and oncogenesregulate AP-1 binding to DNA (Bernstein et al., Science 1989, 244,566-569). Activated AP-1 has been shown to play a role in apoptosis,angiogenesis and metastasis (Kang et al., Am. J. Pathol. 2005, 166(6),1691-1699) and is also involved in many diseases including cancer,diabetes and Alzheimer's disease. AP-1 is also associated with theproduction of metalloproteinases. Collagenases, a class ofmetalloproteinases, are known to contain AP-1 response elements in theirDNA promoters (Kang et al., Am. J. Pathol. 2005, 166(6), 1691-1699). Thecombination of these factors makes AP-1 crucial to many oncogenicprocesses.

AP-1 consists of 18 dimeric combinations of the families Jun (c-Jun,JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) (Young et al.,Trends Mol. Med. 9(1), 36-41 (2003)). Of the dimeric possibilities areJun-Jun homodimers and Jun-Fos heterodimers. Jun dimers bind tightly toAP-1 DNA recognition elements (Angel et al., Cell 49, 729-739 (1987)).Fos-Fos homodimers are unstable and not readily formed but can bind toDNA by forming heterodimers with Jun proteins (Ziegler et al., J. Nutr.2004, 134, 5-10). The most common dimer is a heterodimer consisting ofc-Jun and c-Fos. Also associated with the Jun and Fos families are Jundimerization partners and activating transcription factors (ATF's)(Angel et al., Biochim. Biophys. Acta 1072, 129-157 (1991)).

In normal tissues, the AP-1 component c-Fos is found only in smallconcentrations but cytosolic levels are rapidly increased when the cellis induced by mitogenic stimuli (Muller et al., Nature 1983, 304,454-456). c-Jun, another AP-1 component, plays an important role in theregulation of cellular proliferation (Karin et al., Curr. Opin. CellBiol. 1997, 9(2), 240-246). When c-Jun and c-Fos become unregulated inthe body, abnormal cell proliferation occurs leading to cellulartransformations. c-Jun is known to be essential in tumor promotion inseveral cell lines (Jochum et al., Oncogene 2001, 20(19), 2401-2412;Orlowski et al., Trends Mol. Med. 2002, 8,385-389; Pain, Eur. J.Biochem. 1996, 236, 747-771; Karin et al., Nat. Rev. Cancer 2002, 2(4),301-310; Dhar et al., Mol. Cell. Biochem. 2002, 234-235, 185-193). c-Fosis also involved in the conversion of cells from benign to malignant(Dong et al., Proc. Natl. Acad. Sci. USA 1994, 91, 609-613; Greenhalghet al., Cell Growth Differ. 1995, 6, 579-586) and is essential in tumorprogression (Saez et al., Cell 1995, 82(5), 721-732). In general, theactivation of both NF-κB and AP-1 are required for tumor promotion andprogression.

The AP-1 activation cascade can be induced by TNFα, okadaic acid,12-O-tetradecanoylphorbol-13-acetate (TPA), UV light (Young et al.,Trends Mol. Med. 9(1), 36-41 (2003)), cytokines, mitogens, phorbolesters, growth factors, environmental and occupational particles, toxicmetals, intracellular stresses, bacterial toxins, viral products andionizing radiation (Fontecave et al., FEBS Lett. 421, 277-279 (1998)).In general, AP-1 is activated primarily through mitogen-activatedprotein kinase (MAPK) cascades (Kundu et al., Mutat. Res. 2004, 555,65-80). MAPK's are composed of MAPK itself and MAPK kinase, also calledMAPK-extracellular signal regulated kinase (MEK) (Wilkinson et al.,Genes Dev. 1998, 12, 1391-1397). MAPK's are activated by cytokines,hormones and stress-inducing agents (Blenis, Proc. Natl. Acad. Sci. USA1993, 90(13), 5889-5892). In general, the same factors that stimulateNF-κB also stimulate AP-1.

MAPK or MEK can phosphorylate additional kinases including extracellularregulating kinases (ERK's), c-Jun N-terminal kinase (JNK) and p38 MAPK(Baker et al., Mol. Cell. Biol. 1992, 12(10), 4694-4705; Davis, J. Biol.Chem. 1993, 268(20), 14553-14556). JNK activates the c-Jun protein andERK activates a protein called Elk-1 both by phosphorylation. c-Jun thenbinds to DNA along with an ATF to activate genes that produce more ofthe Jun family in a positive feedback loop (Thevenin et al., J. Biol.Chem. 1991, 266(15), 9363-9366). Elk-1 also binds to DNA with a serumresponse factor (SRF) to activate genes that produce the Fos family. TheJun and Fos protein families are then activated by JNK andc-Fos-regulating kinase (FRK) respectively. The activated families cannow dimerize, bind to DNA and activate gene expression that adverselyaffects cellular processes. A pictorial representation of AP-1activation is shown in FIG. 1B.

AP-1 proteins and their activating kinases are related to NF-κB. AP-1proteins are known to interact with the p65 subunit of NF-κB (Li et al.,Mol. Carcinog. 29(3), 159-169 (2000)). MAPK's are known to phosphorylateI-κB (Adler et al., EMBO J. 18, 1321-1334 (1999)). Because AP-1 andNF-κB responsive genes can promote angiogenesis, cell motility andinvasion, and block apoptotic cell death, activation of these genes andtheir products may result in cancerous or precancerous growth. Moreover,AP-1 and NF-κB responsive genes can promote inflammation, activation ofthese genes and their products may result in greater inflammation indiabetics and others. Therefore, there is a greatly felt need fordevelopment of small molecule inhibitors of AP-1 or NF-κB activation.

Alzheimer's Disease

Alzheimer's disease (AD), the most common cause of dementia in elderlypopulations, currently afflicts almost 5 million people in the U.S., andthis number is estimated to increase to 15 million by 2050. Hebert etal., (2003) Arch Neurol 60, 1119-1122. Most AD is sporadic with multiplerisk factors, while some 10-15% is familial. It is well accepted thatexcessive production or diminished clearance of the Aβ peptide derivedfrom the amyloid precursor protein (APP) is an essential factor in theetiology of AD. This is supported by studies of genetic mutations in APPin experimental animal models of AD as well as from studies of thegenetics of familial AD. Selkoe et al., (2000) Annu Rev Genomics HumGenet 3, 67-99.

There are two major neuropathological signatures of AD: extraneuronalamyloid plaques and neurofibrillary tangles. The plaques primarilyconsist of Aβ aggregates while the tangles consist ofhyperphosphorylated tau protein. The exact mechanism by which theseaggregates cause neuronal cell death remains to be established. However,considerable recent evidence points towards a major role for oligomericforms of Aβ which are neurotoxic and can diffuse. Soluble Aβ is found inCSF of AD patients and correlates better with severity of disease thandoes the quantity of plaques. Kim et al., (2003) FASEB J 17, 118-120;McLean et al., (1999) Ann Neurol 46, 860-866.

There are other common features of AD including the presence of chronicinflammation. The inflammatory response in brain is directed byactivated microglia and reactive astrocytes. In normal brain, microgliaare not activated. Under these conditions, neither pro-inflammatorysignals nor reactive oxygen/nitrogen species (ROS/RNS) are formed.McGeer et al., (2003) Prog Neuropsychopharmacol Biol Psychiatry 2,741-749. However, when microglia become activated in response to variousinsults, there is up-regulation of a number of surface receptors thatpromote phagocytotic activity by microglia. In addition,pro-inflammatory signals are released including interleukin-1β (IL1β)and tumor necrosis factor-α (TNFα) as well as ROS/RNS, thus contributingto the oxidative stress associated with AD. Activated microglia alsoassociate with amyloid plaques. Microglia isolated from AD brain canscavenge Aβ. Rogers et al., (2002) Glia 40, 260-269. The considerableliterature on the role of microglia in AD suggests that activation ofmicroglia may contribute initially to clearance of Aβ aggregates, butthat the chronic activation of microglia observed in AD leads to theneuropathological changes in the AD brain. Griffin et al., (1998) BrainPathol 8, 65-72. Activated microglia also contribute tohyperphosphorylation of tau with development of neurofibrillary tangles,as well as to recruitment of activated astrocytes into the Aβ plaques.Kitazawa et al., (2004) Ann NY Acad Sci 1035, 85-103.

It is now recognized that Aβ can increase the inflammatory response byactivation of microglia and that the inflammatory response cancontribute to Aβ deposition. Consequently there has been interest inhindering microglial activation as an approach to breaking thispathological cycle. Aisen (1997) Gerontology 43, 143-149. Sinceactivation of microglial results in release of ROS/RNS, attention hasfocused on use of anti-oxidants such as vitamin E. There are conflictingreports of the effects of anti-oxidants on development of AD, somesupporting a role for anti-oxidants (Engelhart et al., (2002) JAMA 287,3223-3229) and others not supporting a role (Laurin et al, (2004) Am JEpidemiol 159, 959-967). Activation of microglia increases the oxidativeburden in affected brain regions. However, how significant this increaseis in contributing to neurodegeneration is not known. The field ofanti-oxidant treatment of AD will need further controlled trials toassess this question.

Another area that has produced conflicting reports is the use ofanti-inflammatory drugs, especially non-steroidal anti-inflammatorydrugs (NSAIDS), in treatment of AD. COX-2, the inducible form ofcyclooxygenase found in neurons and other cells and the source ofpro-inflammatory eicosenoids, is up-regulated in AD brains. Yasojima(1999) Brain Res 830, 226-236. Overexpression of human COX-2 in miceresults in age-related cognitive decline as well as neuronal apoptosisand astrocyte activation. Andreasson et al., (2001) J Neurosci 21,8198-8209. The epidemiology studies of use of COX inhibitors (i.e.NSAIDs) by AD patients suggest that NSAID therapy may be useful. McGeeret al., (2003) Prog Neuropsychopharmacol Biol Psychiatry 2, 741-749.However, controlled clinical trials have been disappointing. Theseconflicting results may reflect the fact that the epidemiology studiesbegin with normal subjects and then assess risk of developing diseaseand whether this risk correlates inversely with drug use, whereas theclinical trials begin with subjects who have AD and look for improvementupon treatment. Other studies suggest that only a limited group ofNSAIDs are effective and that these NSAIDs influence multiple targets inaddition to COX-2. Gasparini et al., (2005) Brain Res Rev 48, 400-408.Animal model studies suggest that the dosing level of NSAID that isclinically feasible may not be sufficient to produce a pharmacologicaldose at the sites of plaque formation in AD brains. Cole et al., (2004)Ann NY Acad Sci 1035, 68-84.

Another area of interest in AD drug development focuses on signalingpathways that regulate expression of pro-inflammatory genes. Aβstimulation of microglia results in up-regulation of the expression ofTNFα and IL1 that is at least partly NFκB-dependent. Combs et al.,(2001) J. Neurosci. 21, 1179-1188. IL1 is known to affect the expressionof over 90 genes including those for cytokines, cytokine receptors,tissue remodeling enzymes and adhesion molecules. O'Neill (1995) BiochimBiophys Acta 1266, 31-44. The mechanism for IL1 action involvesactivation of an IL1 receptor-mediated signal transduction pathway whichleads to activation of NFκB. O'Neill et al., (1998) J Leukoc Biol 63,650-657. Thus NFκB is involved both in up-regulation of IL1 and inexpression of the multiple genes regulated by IL1. These observationsmake inhibition of NFκB an attractive target for control ofIL1-responsive genes in brain inflammation.

Diabetes

In 1998, it was suggested that the innate immune system is activated indiabetes, leading to a chronic inflammatory state that contributes tothe disease process (Pickup et al., 1998, Diabetologia 41:1241-1248).More recently, there has been considerable support not only for aninflammatory contribution to diabetes but also to diabetic complications(Navarro et al., 2005, Nephrol Dial Transplant 20:2601-2604;Pillarisetti et al., 2004, Expert Opin Ther Targets 8:401-408).Specifically, pro-inflammatory cytokines play a major role inmicrovascular complications. Endogenous production of TNF-α in vasculartissue is accelerated in diabetes where it contributes to increasedvascular permeability in diabetic neuropathy (Satoh et al., 2003, ExpDiabesity Res 4:65-71). Both TNF-α and IL-1 expression are increased indiabetic retina where chronic low-grade inflammation appears tocontribute to retinopathy (Joussen et al., 2002, FASEB J 16:438-440).Likewise, diabetic nephropathy is associated with expression ofinflammation markers such as CRP, fibrinogen and IL-6, and withincreased expression of adhesion molecules such as ICAM-1, which promoteinflammation by increasing leukocyte adherence and infiltration (DallaVestra et al., 2005, J Am Soc Nephrol 16:S78-S82). The responses tothese pro-inflammatory cytokines are especially prominent in endothelialcells (EC). Moreover, the response of EC to these cytokines commonlyinvolves signaling through transcription factor NF-κB (Mohamed et al.,1999, BioFactors 10:157-167).

Oxidative stress has consistently been shown in experimental models ofdiabetes (Mohamed et al., 1999, BioFactors 10:157-167). Multiplemechanisms are involved that produce oxidative stress in EC in responseto hyperglycemia, including: 1) protein glycosylation leading to AGEthat trigger ROS production upon binding to the AGE receptor (RAGE)(Wautier et al., 2004, Circ Res 95:233-238); 2) glucose auto-oxidation(Ceriello, 1997, Diabet Med 14:S45-S49); 3) accelerated metabolism ofglucose through the aldose reductase/polyol pathway which consumes NADPH(Srivastava et al., 2005, Endocrin Rev 26:380-392); 4) uncoupling ofoxidative phosphorylation and of endothelial NO synthase (eNOS) (Satohet al., 2005, Am J Physiol Renal Physiol 288:F1144-F1152); 5) activationof specific isoforms of PKC (Ahmed et al., 2005, Curr Drug Targets6:487-494); 6) increased flux through the hexosamine pathway (Schleicheret al., 2000, Kidney Int Suppl 77:S13-S18); and 7) exposure toangiotensin II (Yamagishi et al., 2005, FEBS Letters 579:4265-4270).Activation of NF-κB is often observed in response to these stresses. Forexample, exposure of EC to AGE generates ROS through activation of NADPHoxidase which then activates NF-κB followed by up-regulation ofNF-κB-dependent cytokines and adhesion molecules (Wautier et al., 2001,Am J Physiol Endocrinol Metab 280:E685-E694). Angiotensin II can augmentthis process through crosstalk with the AGE-RAGE system, again involvingNF-κB (Yamagishi et al., 2005, FEBS Letters 579:4265-4270). High glucosecan induce EC apoptosis through a PI-3-kinase-regulated expression ofCOX-2; this was shown to involve ROS and the NF-κB-regulated expressionof COX-2 (Sheu et al., 2005, Arterioscler Thromb Vasc Biol 25:539-545).There has been considerable interest in a role for poly(ADP)-ribosepolymerase (PARP) in EC dysfunction. PARP directly interacts with boththe p50 and p65 subunits of NF-κB, suggesting that the role of PARPactivation in diabetic complications is, at least in part, due to itsinteraction with NF-κB (Zheng et al., 2004, Diabetes 53:2960-2967).Glucose-induced activation of NF-κB in EC is prevented by inhibitors ofPKC, suggesting that the role of PKC in triggering the expression ofpro-inflammatory cytokines is through downstream activation of NF-κB(Pieper et al., 1997, J Cardiovasc Pharmacol 30:528-532). There has alsobeen considerable interest in mitochondria-derived ROS (specificallysuperoxide) produced in response to hyperglycemia and the relationshipbetween these ROS and enhanced flux through the polyol pathway and thehexosamine pathway, PKC activation, and intracellular generation of AGE,all of which can be prevented by inhibiting the formation ofmitochondria-derived ROS (Nishikawa et al., 2000, Nature 404:787-790).The activation of these biochemical pathways appears to be due toROS-induced activation of PARP, which results in inactivation ofglyceraldehyde-3-phosphate dehydrogenase and subsequent accumulation ofglycolytic intermediates that promote these pathways (Araujo et al.,2001, Mem Inst Oswaldo Cruz 96:723-728). It is noteworthy thatinhibiting the production of mitochondria-derived ROS also prevents theactivation of NF-κB (Du et al., 2003, J Clin Invest 112:1049-1057),which may be related to the activation status of PARP. Clearly,activation of NF-κB appears to be a general feature of EC that arestressed by factors related to diabetic complications, suggesting acentral role for NF-κB in EC dysfunction, especially as the keyregulator of pro-inflammatory cytokines, adhesion molecules andextracellular matrix components, all of which are major players indiabetic microvascular complications.

The signaling mechanisms involved in inflammation that contributes todiabetes are under investigation, and are described by Wellen et al.(Wellen et al., J. Clin. Invest., 115, 1111-1119). This researchindicates that inflammatory signaling pathways can be activated bymetabolic stress or extracellular signaling molecules, and thatendoplasmic reticulum stress (ER stress) leads to the activation ofinflammatory signaling pathways and thus contributes to insulinresistance. Ozcan et al., Science, 306, 457-461 (2004). For example,several serine/threonine kinases are activated by inflammatory orstressful stimuli that contribute to inhibition of insulin signaling,including c-Jun N-terminal kinase (JNK) and I-κB kinase (IKK). The threemembers of the JNK group of kinases (JNK-1, -2, and -3) belong to theMAPK family and regulate multiple activities, in part through theirability to control transcription by phosphorylating activator protein-1(AP-1). Loss of JNK1 has been shown to prevent the development ofinsulin resistance and diabetes in both genetic and dietary models ofobesity. Hirosumi et al., Nature, 420, 333-336 (2002).

A model of the overlapping metabolic and inflammatory signaling andsensing pathways in adipocytes and macrophages that influence diabetesand inflammation is provided by FIG. 2. As shown in FIG. 2, signals fromvarious mediators converge on the inflammatory signaling pathways,including the kinases JNK and IKK. These pathways lead to the productionof additional inflammatory mediators such as NF-κB and AP-1 throughtranscriptional regulation as well as to the direct inhibition ofinsulin signaling. Opposing the inflammatory pathways aretranscriptional factors from the PPAR and LXR families, which promotenutrient transport and metabolism and antagonize inflammatory activity.

Glutathione S-Transferase

Glutathione S-transferases (GSTs) are a superfamily of enzymesclassified into eight gene families. Many GSTs are also classified asphase II detoxification enzymes that catalyze the conjugation ofglutathione to a wide variety of electrophiles as the first step inelimination of xenobiotics. However, GSTs also exhibit numerousfamily-specific functions, some but not all of which involveglutathione. For example, GSTP1-1, which is the main member of the “pi”family and is the most widely distributed GST, is important as both adetoxification enzyme and in signal regulation through itsprotein-protein interactions with c-Jun N-terminal kinase (JNK), akinase that is important in the stress response and apoptosis. Thus,up-regulation of GSTP1-1 serves to protect cells from apoptosis-inducingstress by inhibiting JNK. Notably, the promoter for GSTP1-1 containsNFκB-binding sites. It is important to understand that oxidative stressleads to modification of critical cysteine residues in GSTP1-1,resulting in the release of JNK and initiation of apoptosis. Therefore,it is known that tumors that over-express GSTP1-1 are resistant tostress-induced apoptosis, and the presence of GSTP1-1 assists in theprevention of apoptosis.

Glutathione S-Transferase P1-1 (GSTP1-1) thus has two distinct functionswhich contribute to the survival of cancer cells. First, GSTP1-1detoxifies xenobiotic electrophiles, including some cancer drugs, bycatalyzing the conjugation of glutathione, thereby contributing to drugresistance. It is involved in eliminating toxic molecules from the cellincluding drugs that are supposed to be assisting the cell in fightingdiseases, and has been implicated in the development of drug resistancein a variety of cancers. Elevated levels of GSTP1-1 are found innumerous cancer cell lines and tumors, including, among others, breastcancers, prostate cancers, and leukemias that are resistant to a rangeof anti-cancer drugs. It is known in the art that GSTP1-1 positivebreast tumors are more aggressive than GSTP1-1 negative tumors and havea poorer prognosis. For example, the MCF7 breast cancer cell line, whichis a GSTP1-1 expressing line, was shown to develop resistance to anumber of drugs when the cells were transfected with GSTP1-1. It is alsoknown in the art that ovarian cancer cell lines that over-expressGSTP1-1 are resistant to taxol and doxorubicin. In fact, GSTP1-1 hasalso been used as a prognostic tool in invasive breast cancer.Over-expression of GSTP1-1 has been shown to be a marker of poor outcomein breast cancer and advanced non-Hodgkin's lymphoma. And second,because GSTP1-1 also inhibits the pro-apoptotic factor c-Jun N-terminalkinase (JNK), it promotes the pro-survival state.

A number of studies support the idea that inhibitors of GSTP1-1 may havetherapeutic potential in the treatment of cancer. If GSTP1-1 can beinhibited, then known cancer therapeutics would not be eliminated fromthe cell. In one study, inhibition of GSTP1-1 by the glutathioneconjugate of doxorubicin induces apoptosis in rat hepatoma cells. Also,ethacrynic acid, a broad-spectrum inhibitor of glutathioneS-transferases, provides a therapeutic advantage when combined withother agents. Ethacrynic acid, however, is a potent diuretic; this alongwith its lack of GST isozyme selectivity precludes the development ofethacrynic acid as an anti-cancer therapeutic. A number ofpeptidomimetic inhibitors that are selective for GSTP1-1 are in variousstages of development, including one in Phase III for non-small celllung cancer and ovarian cancer.

Curcumin

Nontraditional or alternative medicine is becoming an increasinglyattractive approach for the treatment of various inflammatory disorders.Among these alternative approaches is the use of food derivatives, whichhave the advantage of being relatively nontoxic. A number of dietarycompounds such as flavonoids and curcumin block activation of NFkB(Yamamoto et al., J. Clin. Invest., 107, 135 (2001); Bharti et al.,Blood 101, 1053 (2003)). Curcumin is a non-nutritive, non-toxicpolyphenol natural product found in turmeric, a spice that has been usedfor centuries in India and elsewhere as an herbal medicinal treatment ofwounds, jaundice, and rheumatoid arthritis (Ammon et al., Planta Med.,57, 1 (1991)). Curcumin is the major constituent of turmeric powderextracted from the rhizomes of the plant Curcuma longa L found in southand southeast tropical Asia (Govindaraja, V. S., Crit. Rev. Food Sci.Nutri. 12:199 (1980)). In the countries of its origin, turmeric has alsobeen used for centuries as a traditional medicine to treat inflammatorydisorders. Scientists have subsequently demonstrated theanti-inflammatory properties of curcumin (Ammon et al., Planta Med. 57:1(1991). Curcumin also exhibits potent anti-oxidant activity, whichdepends upon the presence of phenolic groups in the aryl rings (Baldwin,A. S. J. Clin. Invest. 107:241 (2001)). In traditional Indian medicine,curcumin has been used to treat a host of ailments through topical, oraland inhalation administration, and has recently been found safe in sixhuman trials at oral loads up to 8 grams/day for 6 months. Chainani-Wu(2003) J Altern Complement Med 9, 161-168. Most of the clinical trialsof curcumin pertain to its anti-tumor activity in colon, skin, stomach,duodenal, soft palate and breast cancers. However, the mechanism ofaction for curcumin is not well understood.

Curcumin derivatives have been shown to provide antitumor activity. Forexample, the antitumor activity of curcumin derivatives is described inU.S. patent application Ser. No. 11/057,636, entitled “Method andCompounds for Cancer Treatment Utilizing NFkB as a Direct or UltimateTarget for Small Molecule Inhibitors,” filed Feb. 14, 2005, by VanderJagt et al. and incorporated herein by reference, and U.S. patentapplication Ser. No. 11/373,444, entitled “Cancer Treatment UsingCurcumin Derivatives,” filed Mar. 10, 2006, also by Vander Jagt et al.and incorporated herein by reference.

Curcumin is a natural chemoprotective agent that elevates the activitiesof Phase 2 detoxification enzymes, while inhibiting procarcinogenactivating Phase 1 enzymes. It decreases expression of severalproto-oncogenes including c-jun, c-fos, and c-myc, and of particularinterest, it suppresses the activation of NFκB. Related to this,curcumin has also been shown to induce apoptosis in several tumor celllines. In addition to the down-regulation of uPA by dominant negativeinhibitors of NFκB, numerous other factors, including VEGF, IL-8, andMMP-9 that contribute to angiogenesis, invasion, and metastasis aredown-regulated by dominant negative inhibitors of NFκB. Likewise,curcumin inhibits angiogenesis in vivo. Curcumin can be viewed as a leadcompound that inhibits metastasis and promotes apoptosis. Otherantiangiogenic properties of curcumin are also known. Shim et al. haveshown that curcumin causes the irreversible inhibition ofCD13/aminopeptidase N, a membrane-bound, zinc-dependentmetalloproteinase that plays a key role in tumor invasion andangiogenesis. Shim et al., “Irreversible inhibition ofCD13/aminopeptidase N by the antigenic agent curcumin”, Chem. Biol.10(8): 695-704 (August 2003).

Curcumin is known to inhibit the formation of Jun-Fos heterodimers inTPA induced cells and curcumin analogs are known to be up to 90 timesmore potent than curcumin (Hahm et al., Cancer Lett. 184, 89-96 (2002).It is also known that besides curcumin (turmeric), several naturalproducts including resveratrol (peanuts and grape skins) (Manna et al.,J. Immunol. 164, 6509-6519 (2000)), silymarin (artichoke) (Manna et al.,J. Immunol. 163(12), 6800-6809 (1999)), oleandrin (Manna et al., CancerRes. 60, 3838-3847 (2000)) and several compounds isolated from bothgreen and black tea leaves (Chung et al., Cancer Res. 59, 4610-4617(1999)) inhibit the AP-1 activation cascade. It is possible thatcurcumin analogs exhibit their activities on JNK since it is known thatboth silymarin (Manna et al., J. Immunol., 163(12), 6800-6809 (1999))and oleandrin (Manna et al., Cancer Res. 60, 3838-3847 (2000)) inhibitJNK activity.

In addition, curcumin exhibits anti-inflammatory activity and is apotent anti-oxidant and free radical scavenger. Leu et al., (2002) CurrMed Chem Anti-Canc Agents 2, 357-370. In APP-overexpressing transgenicmice, curcumin reduced levels of oxidized proteins and inflammatorycytokine IL1 (Lim et al., (2001) J Neurosci 2, 8370-8377), thus offeringa potential therapy against microglial activation in patients withAlzheimer's disease. Curcumin has additional activities of interest: itlimits the progression of renal lesions in the STZ-diabetic rat model(Suresh Babu et al., (1998) Mol Cell Biochem 181, 87-96), andameliorates oxidative stress-induced renal injury in mice (Okada et al.,(2001) J Nutr 131, 2090-2095). Consequently, there has been extensiveinterest in the anti-oxidant properties of curcumin and the possibilitythat many of its biological activities are derived from its anti-oxidantproperties. Balasubramanyam et al., (2003) J Biosci 28, 715-721.

Curcumin also inhibits the activation of NFκB (Bharti et al., (2003)Blood 101, 1053-1062), which may explain its anti-inflammatoryproperties. Curcumin was shown to attenuate the plasma inflammatorycytokine surge and cardiomyocyte apoptosis following cardiacischemia/reperfusion in experimental animals by inhibiting activation ofNFκB. Yeh et al., (2005) J Surg Res 125, 109-110. Curcumin suppressedNOS induction in LPS-stimulated macrophages by inhibiting the activationof NFκB. Pan et al., (2000) Biochem Pharmacol 60, 1655-1676. Likewise,curcumin inhibited mitogen stimulation of lymphocyte proliferation byinhibiting activation of NFκB. Ranjan et al., (2004) J Surg Res 121,171-177. Of particular interest is the report that curcumin inhibits theactivation of NFκB in BV2 microglia cells (Kang et al, (2004) JPharmacol Sci 94, 325-328). The limited bioavailability of curcumin(Garcea et al., (2004) Br J Cancer 90, 1011-1015) suggests that clinicaluse of this natural product will be limited and points to the need todevelop curcumin analogs with improved properties including improvedbioavailability.

It was reported that curcumin inhibits TNF-α-induced NF-κB activation inhuman myelomonoblastic leukemia cells and phorbol ester-inducedc-Jun/AP-1 activation in mouse fibroblast cells (Singh et al., J. Biol.Chem. 270:24995 (1995); Huang et al., Proc. Natl. Acad. Sci. USA 88:5292(1991). The molecular mechanism for NF-κB inhibition by curcumin wasunclear, but involved inhibition of I-κB degradation (Kumar et al.,Biochem. Pharmacol. 55:775 (1998). More recent work has demonstratedthat curcumin blocks intestinal endothelial cell gene expression byinhibiting the signal leading to IKK activation without directlyinterfering with NIK or IKK, and that blockade of IKK activation causesinhibition of I-κB phosphorylation/degradation and NF-κB activation(Jobin et al., J. Immunol. 163, 3474-83 (1999)).

The anti-inflammatory properties of curcumin and its ability to inhibitthe immune response upon exposure to a variety of external stimuli may,at least in part, result from inhibition of the activation of NF-κB bythese external signals, since many of the genes that are implicated inthe immune/inflammatory response are up-regulated by NFκB. For example,curcumin inhibits the LPS-induced production of IL-1β and TNFα (Chan, M.M. Biochem. Pharmacol. 49, 1551 (1995)) and the IL-1β-induced expressionof IL-2 (Chaudhary, L. R.; Avioli, L. V. J. Biol. Chem. 271, 16591(1996)), as well as the TNFα-induced expression of ICAM-1, VCAM-1 andE-selectin (Gupta, B.; Ghosh, B. Int. J. Immunopharmacol. 21, 745(1999)). NF-κB is implicated in these signaling pathways (Wang et al.,Cytokine 29, 245 (2005); Krunkosky et al., Free Radical Biol. Med. 35,1158 (2003)). However, curcumin has also been shown to be a directinhibitor of enzymes that are important in the inflammatory response,including lipoxygenase and cyclo-oxygenase (Skrzypczak-Jankun et al., J.Int. J. Mol. Med. 6, 521 (2000)).

Further, curcumin has been shown to have possible application in thetreatment of cystic fibrosis defects caused by mutations in the gene forthe cystic fibrosis transmembrane conductance regulator (CFTR),particularly for A508 mutations. Egan, et al., “Curcumin, A MajorConstituent of Turmeric, Corrects Cystic Fibrosis Defects”, Science,304: 600-602 (23 Apr. 2004).

The large consumption of curcumin by the Indian population may helpexplain their relatively low (4 times less) incidence of Alzheimer'sdisease compared to the U.S. population. Chandra et al., (2001)Neurology 57, 985-989. Although no systematic trials have been preformedusing curcumin in India, recent studies have provided valuable insightson curcumin's role in Alzheimer's disease. Yang et al., (2005) J BiolChem 280, 5892-5901; Ono et al., (2004) J Neurosci Res 75, 742-750.Curcumin was shown to inhibit the formation of Aβ oligomers and fibrilsin vitro and reduce Aβ amyloid burden in vivo. Specifically, Ono et al.have indicated that curcumin inhibits the accumulation of amyloidβ-peptide (Aβ) and the formation of β-amyloid fibrils (fAβ) from Aβ anddestabilizes preformed fAβ. Ono et al., “Curcumin Has PotentAnti-Amyloidogenic Effects for Alzheimer's β-Amyloid Fibrils In Vitro”,J. Neuroscience Res., 75: 742-750 (2004). Importantly, curcuminadministered by intravenous (i.v.) injection lowered Aβ deposition inaged APP(Swedish)-transgenic mice (Tg2576), clearly demonstrating itsability to cross the blood-brain barrier in sufficient quantities toreduce amyloid burden. Curcumin is structurally similar to otherinhibitors of Aβ aggregation such as Congo Red and Chrysamine G.

Thus, NFκB and its upstream regulators, as well as AP-1 and GSTP1-1,present inviting targets for development of anti-inflammatory drugs, andcurcumin represents a promising lead compound. Analogues of curcuminthat function as small molecules inhibitors of NFκB, AP-1 and GSTP1-1activation are highly desirable for the treatment of diseases withinflammatory symptoms or components such as Alzheimer's disease,diabetes, cystic fibrosis and cancer, and also as assistive or adjuvantagents in the chemotherapeutic treatment of cancer.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of treating a subjectafflicted with a disease, wherein the method includes administering tothe subject a therapeutically effective amount of a curcumin derivative.

In one embodiment of the method of the invention, the curcuminderivative is a compound having Formula I (Ar¹—L—Ar²), wherein Ar¹ andAr² are each independently aryl groups, and L represents a divalentlinking group.

In one embodiment of the compound of Formula I, Ar¹ is a phenyl groupaccording to Formula II:

and/or Ar² is a phenyl group according to Formula III:

wherein each of R¹-R¹⁰ is independently selected from the groupconsisting of hydrogen, hydroxyl, methyl, methoxyl, dimethylamine,trifluoromethyl, chloro, fluoro, acetoxyl, cyano, and carboxymethyl.Alternatively or additionally, either or both of Ar¹ and Ar² areindependently heteroaryl groups.

In another embodiment of the method of the invention, the curcuminderivative is a compound having Formula IV (Ar¹—L—R¹¹), wherein Ar¹ isan aryl group, L represents a divalent linking group, and R¹¹ is analkyl group, a heterocyclic group, or a hydrogen. In one embodiment, Ar¹is a phenyl group according to Formula II, wherein each of R¹-R⁵ isindependently selected from the group consisting of hydrogen, hydroxyl,methyl, methoxyl, dimethylamine, trifluoromethyl, chloro, fluoro,acetoxyl, cyano, and carboxymethyl.

The divalent linking group L preferably includes an alkylene or analkenylene including 3, 4, 5, 6, or 7 backbone carbon atoms, wherein oneor more of the backbone carbon atoms form part of a carbonyl orsecondary alcohol. The linking group L may be saturated or unsaturated;preferably, L contains at least one unsaturated carbon-carbon bond.

In a preferred embodiment, L is an alkylene or an alkenylene selectedfrom the group consisting of: —CH═CH—CHO—, —CH═CH—(CO)—CH═CH—,—CH₂—CH₂—(CO)—CH₂—CH₂—, —CH₂—CH₂—CH(OH)—CH₂—CH₂—,

—CH═CH—(CO)—CR—C(OH)—CH═CH—, —CH═CH—(CO)—CR₂—(CO)—CH═CH—, and—CH═CH—(CO)—CH—C(OH)—CH═CH—; R is an alkyl or aryl group including 10carbon atoms or less.

The curcumin derivative of the invention may be administered as apharmaceutical composition, optionally containing a pharmaceuticallyacceptable carrier.

The method of the invention is useful for treating any disease orcondition characterized by inflammation, including Alzheimer's disease,diabetes (particularly type 2 diabetes), cancer or a precancerouscondition (e.g., dysplasia or hyperplasia), cystic fibrosis, rheumatoidarthritis, asthma, inflammatory bowel disease, ulcerative colitis,atherosclerosis and stroke. A subject afflicted with diabetes who istreated in accordance with the invention may exhibit endothelialdysfunction by one or more endothelial cells that express activatedNF-κB or AP-1. It should be understood that the method of the inventionis generally useful for treating any disease or condition that can beameliorated by inhibiting the activity of NFκB, AP-1 and/or GSTP1-1.

In some embodiments of the method of treating a subject with Alzheimer'sdisease, the composition inhibits amyloid plaque formation. In otherembodiments, the composition inhibits aggregation of a plurality of Aβpeptides. In additional embodiments, the composition inhibitsoligomerization of a plurality of Aβ peptides. In further embodiments,the composition decreases the cytotoxicity of an Aβ peptide aggregate.In yet further embodiments, the composition decreases activation of aglial cell by an Aβ peptide aggregate.

The curcumin derivatives provided herein optionally inhibit the activityof the enzymes AP-1, NF-κB and/or GSTP1-1. Inhibition of enzyme activitymay be observed or demonstrated by in vitro assays, in vivo, or both.Inhibition of enzyme activity may decrease inflammation, insulinresistance and/or render a cancer cell more susceptible to achemotherapeutic agent.

In embodiments of the invention that involve the treatment of cancer ora precancerous condition, the curcumin derivative may be administered tothe subject either alone or in combination with one or more other cancerdrugs (e.g., chemotherapeutic agents), for example in an assistive oradjuvant capacity. The curcumin derivative may be administered before,concurrent with, or after the administration of the other cancerdrug(s).

In another aspect, the present invention provides methods foridentifying an therapeutic curcumin derivative that includes contactinga cell containing NF-κB, AP-1 and/or GSTP1-1 with a curcumin derivative,contacting the cell with an activator of NF-κB, AP-1 and/or GSTP1-1, anddetermining the effect of the curcumin derivative on cell activation bythe activator, wherein a curcumin derivative that reduces cellactivation is identified as a therapeutic curcumin derivative. Exemplaryactivators include TNF-α or IL-1. In a further embodiment, the cell isan adipocyte or endothelial cell.

In another aspect, the invention provides a method for identifying atherapeutic curcumin derivative that includes contacting a brain cellcomprising an inflammation activator with a curcumin derivative anddetermining the effect of the curcumin derivative on activation of thebrain cell by the inflammation activator. A curcumin derivative thatreduces brain cell activation when tested by this method is identifiedas a therapeutic curcumin derivative. The inflammation activator of thismethod may include NF-κB or AP-1. In one or more embodiments, the braincell is a glial cell.

In another aspect, the invention provides a method for identifying atherapeutic curcumin derivative that includes contacting a solutionincluding an Aβ peptide with a curcumin derivative and determining theeffect of the curcumin derivative on aggregation by the Aβ peptide. Acurcumin derivative that reduces aggregation of the Aβ peptide isidentified as a therapeutic curcumin derivative. In one ore moreembodiments, effect of the curcumin derivative on aggregation by the Aβpeptide is determined by an immunological assay.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application with color drawingswill be provided by the Office upon request and payment of the necessaryfee.

FIG. 1A is a pictorial representation of the NF-κB activation cascade.

FIG. 1B is a pictorial representation of the AP-1 activation cascade.

FIG. 2 is a pictorial representation of the inflammatory and metabolicpathways affecting insulin resistance and diabetes.

FIG. 3A is a bar graph showing the activities of curcumin analogsincluding 7-carbon linker groups in the TRAP assay;

FIG. 3B is a bar graph showing the activities of curcumin analogsincluding 5-carbon linker groups in the TRAP assay;

FIG. 3C is a bar graph showing the activities of curcumin analogsincluding 3-carbon linker groups in the TRAP assay;

FIG. 4A is a bar graph showing the activities of curcumin analogsincluding 7-carbon linker groups in the FRAP assay;

FIG. 4B is a bar graph showing the activities of curcumin analogsincluding 5-carbon linker groups in the FRAP assay;

FIG. 4C is a bar graph showing the activities of curcumin analogsincluding 3-carbon linker groups in the FRAP assay;

FIG. 5A is a bar graph showing the activities of curcumin analogsincluding 7-carbon linker groups as inhibitors of the activation ofNF-κB by TNFα;

FIG. 5B is a bar graph showing the activities of curcumin analogsincluding 5-carbon linker groups as inhibitors of the activation ofNF-κB by TNFα;

FIG. 5C is a bar graph showing the activities of curcumin analogsincluding 3-carbon linker groups as inhibitors of the activation ofNF-κB by TNFα;

FIG. 6A is a graph showing an IC₅₀ plot of varying doses of curcuminagainst inhibition of NF-κB activity;

FIG. 6B is a graph showing an IC₅₀ plot of varying doses of analog 31against inhibition of NF-κB activity;

FIG. 6C is a graph showing an IC₅₀ plot of varying doses of analog 29against inhibition of NF-κB activity;

FIG. 6D is a graph showing an IC₅₀ plot of varying doses of analog 38aagainst inhibition of NF-κB activity;

FIG. 6E is a graph showing an IC₅₀ plot of varying doses of analog 20qagainst inhibition of NF-κB activity;

FIG. 6F is a graph showing an IC₅₀ plot of varying doses of analog 20wagainst inhibition of NF-κB activity;

FIG. 6G is a graph showing an IC₅₀ plot of varying doses of analog 20agagainst inhibition of NF-κB activity;

FIG. 6H is a graph showing an IC₅₀ plot of varying doses of analog 20magainst inhibition of NF-κB activity;

FIG. 6I is a graph showing an IC₅₀ plot of varying doses of analog 6aagainst inhibition of NF-κB activity;

FIG. 6J is a graph showing an IC₅₀ plot of varying doses of analog 20vagainst inhibition of NF-κB activity;

FIG. 6K is a graph showing an IC₅₀ plot of varying doses of analog 9aagainst inhibition of NF-κB activity;

FIG. 6L is a graph showing an IC₅₀ plot of varying doses of analog 20aagainst inhibition of NF-κB activity;

FIG. 7 is a computer-generated image representing NF-κB (1IKN) bound toIκB;

FIG. 8 is a computer-generated image representing NF-κB (1IKN) with IκBremoved;

FIG. 9 is a computer-generated image representing the front face ofNF-κB (1IKN) with bound analogs;

FIG. 10 is a computer-generated image representing curcumin bound toNF-κB (1IKN);

FIG. 11 is a computer-generated image representing the opposite face ofNF-κB (1IKN) with bound analogs;

FIG. 12 is a computer-generated image representing NF-κB (1IKN) with MESand bound analogs;

FIG. 13 is a computer-generated image representing curcumin bound toNF-κB (1IKN) with MES;

FIG. 14 is a computer-generated image representing NF-κB (1SVC) bound toDNA;

FIG. 15 is a computer-generated image representing NF-κB (1SVC) with DNAremoved;

FIG. 16 is a computer-generated image representing NF-κB (1SVC) withbound analogs;

FIG. 17 is a computer-generated image representing curcumin bound toNF-κB (1SVC);

FIG. 18 is a computer-generated image representing the opposite face ofNF-κB (1SVC) with bound analogs; and

FIG. 19 is a computer-generated image representing NF-κB (1SVC) with MESand bound analogs.

FIG. 20A is a bar graph showing the activities of curcumin analogsincluding 7-carbon analogs active in the AP-1 assay.

FIG. 20B is a bar graph showing the activities of curcumin analogsincluding 5-carbon analogs active in the AP-1 assay.

FIG. 20C is a bar graph showing the activities of curcumin analogsincluding 3-carbon analogs active in the AP-1 assay.

FIG. 21A is a bar graph showing the activities of curcumin analogsincluding 7-carbon analogues in the AP-1 assay.

FIG. 21B is a bar graph showing the activities of curcumin analogsincluding 5-carbon analogues in the AP-1 assay.

FIG. 21C is a bar graph showing the activities of curcumin analogsincluding 3-carbon analogues in the AP-1 assay.

FIG. 22A is a graph showing an IC₅₀ plot of varying doses of analog 20magainst inhibition of AP-1 activity;

FIG. 22B is a graph showing an IC₅₀ plot of varying doses of analog 31against inhibition of AP-1 activity;

FIG. 22C is a graph showing an IC₅₀ plot of varying doses of analog 20oagainst inhibition of AP-1 activity;

FIG. 22D is a graph showing an IC₅₀ plot of varying doses of analog 9aagainst inhibition of AP-1 activity;

FIG. 22E is a graph showing an IC₅₀ plot of varying doses of analog 6aagainst inhibition of AP-1 activity;

FIG. 22F is a graph showing an IC₅₀ plot of varying doses of analog 20dagainst inhibition of AP-1 activity;

FIG. 22G is a graph showing an IC₅₀ plot of varying doses of analog 20cagainst inhibition of AP-1 activity;

FIG. 22H is a graph showing an IC₅₀ plot of varying doses of analog 38aagainst inhibition of AP-1 activity;

FIG. 22I is a graph showing an IC₅₀ plot of varying doses of analog 29against inhibition of AP-1 activity;

FIG. 22J is a graph showing an IC₅₀ plot of varying doses of analog 20agagainst inhibition of AP-1 activity;

FIG. 22K is a graph showing an IC₅₀ plot of varying doses of analog 20qagainst inhibition of AP-1 activity;

FIG. 22L is a graph showing an IC₅₀ plot of varying doses of curcuminagainst inhibition of AP-1 activity;

FIG. 23 is a computer-generated image representing AP-1 bound to DNA.

FIG. 24 is a computer-generated image representing AP-1 with DNAremoved.

FIG. 25 is a computer-generated image representing the front face ofAP-1 with analogs.

FIG. 26 is a computer-generated image representing the opposite face ofAP-1 with analogs.

FIG. 27 is a computer-generated image representing AP-1 with MES andanalogs.

FIG. 28 is a computer generated pharmacophore model with the structureof curcumin superimposed on the model.

FIG. 29 is a graph showing the high reactivity of curcumin withL-cysteine.

FIG. 30 pictorially represents the design of 293T cells for highthroughput screening using flow cytometry.

FIG. 31 shows the analysis of p53 activity in HeLa cells transduced withdifferent TR constructs as a sample of the use of flow cytometry forscreening.

FIG. 32 shows α (ADAM), β (BACE), and γ secretase cleavage sites in theAmyloid Precursor Protein. Proteolytic cleavage of APP by β- andγ-secretases results in Aβ peptide formation. Shaded, transmembranedomain.

FIG. 33 is a graphical representation of the reactivity of curcumin withL-cysteine. Curcumin (20 μM) was incubated with L-cysteine at theindicated concentrations and absorbance (425 nm) was measured up to 60minutes. Natural log values were determined based on 1 st orderkinetics.

FIG. 34 shows that curcumin does not aggregate in an aqueous environmentat concentrations that are effective in preventing Aβ oligomerization.Curcumin was diluted to the indicated concentrations in phosphatebuffered saline, pH 7.0. Absorbance (425 nm) was measured and analyzedby linear regression analysis.

FIG. 35 shows the effect of curcumin and curcumin-based analogs onAβ(1-40) peptide oligomerization. Biotinylated Aβ(1-40) peptide wasdiluted from 5 mg/ml DMSO stock to 20 μg/ml in phosphate bufferedsaline, pH 6.0, and incubated with the indicated concentrations ofcurcumin or curcumin-analog for 48 h at 37° C. Reactions were mixed withTricine sample buffer (no heating) and resolved by 10-20% Tris-Tricinegel electrophoresis. Following transfer to PVDF membranes, boundmaterial was probed with streptavidin-HRP (1 μg/ml) and visualized bychemiluminescence detection.

FIG. 36 shows a structural comparison between curcumin and illustrativecurcumin derivatives.

FIG. 37 shows synthesis of illustrative enone analogs of curcumin.

FIG. 38 shows curcumin analogs, as well as estimated binding constants.

FIG. 39 shows GSTP1-1 inhibitory activity of a curcumin analog.

FIG. 40 shows GSTP1-1 inhibitory activity of a curcumin analog.

FIG. 41 shows the activity of 7-carbon analogues in the GSTP1-1 assay.

FIG. 42 shows the activity of 5-carbon analogues in the GSTP1-1 assayFIG. 43 shows the activity of 3-carbon analogues in the GSTP1-1 assay

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. Unless otherwise specified, “a,” “an,” “the,” and“at least one” are used interchangeably and mean one or more than one.

The terms “comprising” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

As used herein, the term “organic group” is used for the purpose of thisinvention to mean a hydrocarbon group that is classified as an aliphaticgroup, cyclic group, or combination of aliphatic and cyclic groups(e.g., alkaryl and aralkyl groups). In the context of the presentinvention, suitable organic groups for curcumin derivatives of thisinvention are those that do not interfere with the curcumin derivativestherapeutic activity. In the context of the present invention, the term“aliphatic group” means a saturated or unsaturated linear or branchedhydrocarbon group. This term is used to encompass alkyl, alkenyl, andalkynyl groups, for example.

As used herein, the terms “alkyl”, “alkenyl”, and the prefix “alk-” areinclusive of straight chain groups and branched chain groups and cyclicgroups, e.g., cycloalkyl and cycloalkenyl. Unless otherwise specified,these groups contain from 1 to 20 carbon atoms, with alkenyl groupscontaining from 2 to 20 carbon atoms. In some embodiments, these groupshave a total of at most 10 carbon atoms, at most 8 carbon atoms, at most6 carbon atoms, or at most 4 carbon atoms. Cyclic groups can bemonocyclic or polycyclic and preferably have from 3 to 10 ring carbonatoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl,cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstitutedbornyl, norbornyl, and norbornenyl.

The term “heterocyclic” includes cycloalkyl or cycloalkenyl non-aromaticrings or ring systems that contain at least one ring heteroatom (e.g.,O, S, N).

Unless otherwise specified, “alkylene” and “alkenylene” are the divalentforms of the “alkyl” and “alkenyl” groups defined above. The terms,“alkylenyl” and “alkenylenyl” are used when “alkylene” and “alkenylene”,respectively, are substituted. For example, an arylalkylenyl groupcomprises an alkylene moiety to which an aryl group is attached.

The term “haloalkyl” is inclusive of groups that are substituted by oneor more halogen atoms, including perfluorinated groups. This is alsotrue of other groups that include the prefix “halo-”. Examples ofsuitable haloalkyl groups are chloromethyl, trifluoromethyl, and thelike. Halogens are elements including chlorine, bromine, fluorine, andiodine.

The term “aryl” as used herein includes monocyclic or polycyclicaromatic hydrocarbons or ring systems. Examples of aryl groups includephenyl, naphthyl, biphenyl, fluorenyl and indenyl. Aryl groups may besubstituted or unsubstituted. Aryl groups include aromatic annulenes,fused aryl groups, and heteroaryl groups. Aryl groups are also referredto herein as aryl rings.

Unless otherwise indicated, the term “heteroatom” refers to the atoms O,S, or N.

The term “heteroaryl” includes aromatic rings or ring systems thatcontain at least one ring heteroatom (e.g., O, S, N). In someembodiments, the term “heteroaryl” includes a ring or ring system thatcontains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O,S, and/or N as the heteroatoms. Suitable heteroaryl groups includefuryl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl,triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl,thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl,pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl,naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl,pyrazinyl, 1-oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl,oxadiazolyl, thiadiazolyl, and so on.

The terms “arylene” and “heteroarylene” are the divalent forms of the“aryl” and “heteroaryl” groups defined above. The terms “arylenyl” and“heteroarylenyl” are used when “arylene” and “heteroarylene”,respectively, are substituted. For example, an alkylarylenyl groupcomprises an arylene moiety to which an alkyl group is attached.

The term “fused aryl ring” includes fused carbocyclic aromatic rings orring systems. Examples of fused aryl rings include benzo, naphtho,fluoreno, and indeno.

The term “annulene” refers to aryl groups that are completely conjugatedmonocyclic hydrocarbons. Annulenes have a general formula of C_(n)H_(n),where n is an even number, or C_(n)H_(n+1), where n is an odd number.Examples of annulenes include cyclobutadiene, benzene, andcyclooctatetraene. Annulenes present in an aryl group will typicallyhave one or more hydrogen atoms substituted with other atoms such ascarbon.

When a group is present more than once in any formula or schemedescribed herein, each group (or substituent) is independently selected,whether explicitly stated or not. For example, for the formula —C(O)—NR₂each of the two R groups is independently selected.

As a means of simplifying the discussion and the recitation of certainterminology used throughout this application, the terms “group” and“moiety” are used to differentiate between chemical species that allowfor substitution or that may be substituted and those that, in theparticular embodiment of the invention, do not so allow for substitutionor may not be so substituted. Thus, when the term “group” is used todescribe a chemical substituent, the described chemical materialincludes the unsubstituted group and that group with nonperoxidic O, N,S, Si, or F atoms, for example, in the chain as well as carbonyl groupsor other conventional substituents. Where the term “moiety” is used todescribe a chemical compound or substituent, only an unsubstitutedchemical material is intended to be included. For example, the phrase“alkyl group” is intended to include not only pure open chain saturatedhydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like, but also alkyl substituents bearing furthersubstituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl,halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, “alkyl group”includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls,hydroxyalkyls, sulfoalkyls, etc. On the other hand, the phrase “alkylmoiety” is limited to the inclusion of only pure open chain saturatedhydrocarbon alkyl substituents, such as methyl, ethyl, propyl,tert-butyl, and the like.

The invention is inclusive of the compounds described herein (includingintermediates) in any of their pharmaceutically acceptable forms,including isomers (e.g., diastereomers and enantiomers), tautomers,salts, solvates, polymorphs, prodrugs, and the like. In particular, if acompound is optically active, the invention specifically includes eachof the compound's enantiomers as well as racemic mixtures of theenantiomers. It should be understood that the term “compound” includesany or all of such forms, whether explicitly stated or not (although attimes, “salts” are explicitly stated).

“Treat”, “treating”, and “treatment”, etc., as used herein, refer to anyaction providing a benefit to a patient at risk for or afflicted with adisease, including improvement in the condition through lessening orsuppression of at least one symptom, delay in progression of thedisease, prevention or delay in the onset of the disease, etc.

Treatment, as used herein, encompasses both prophylactic and therapeutictreatment. Curcumin derivatives of the invention can, for example, beadministered prophylactically to a mammal in advance of the occurrenceof disease. Prophylactic administration is effective to decrease thelikelihood of the subsequent occurrence of disease in the mammal, ordecrease the severity of disease that subsequently occurs.Alternatively, curcumin derivatives of the invention can, for example,be administered therapeutically to a mammal that is already afflicted bydisease. In one embodiment of therapeutic administration, administrationof the curcumin derivatives is effective to eliminate the disease; inanother embodiment, administration of the curcumin derivatives iseffective to decrease the severity of the disease or lengthen thelifespan of the mammal so afflicted.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

“Inhibit” as used herein refers to the partial or complete eliminationof a potential effect, while inhibitors are compounds that have theability to inhibit.

The present invention provides methods for the use of curcuminderivatives to treat disease in a subject. The present invention alsoprovides methods for identifying and preparing curcumin derivatives

Curcumin (diferuloylmethane,1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is asymmetrical diphenolic dienone. It exists in solution as an equilibriummixture of the symmetrical dienone (diketo) and the keto-enol tautomer;the keto-enol form is strongly favored by intramolecular hydrogenbonding.

Curcumin contains two aryl rings separated by an unsaturated sevencarbon spacer having two carbonyls. The aryl rings of curcumin contain ahydroxyl group in the para position and a methoxy group in the metaposition.

Curcumin Derivatives

Curcumin derivatives are expected to be beneficial for use in thetreatment methods of the invention. The term “curcumin derivative” isused interchangeably with the term “curcumin analog” and “curcuminanalogue” (alternative spelling) and includes, for example, curcuminderivatives, analogs, curcuminoids and chalcones. In one embodiment, thecurcumin derivative includes first and second aryl groups covalentlyattached by way of a spacer, also referred to herein as a linker or alinking group. In another embodiment, the second aryl group is absent,such that the curcumin derivative contains a first aryl group and thespacer but no second aryl group at the distal end of the spacer.Optionally, the first and/or second aryl group is a heteroaryl group.The first and second aryl groups may be independently substituted orunsubstituted.

Representative curcumin derivatives are described herein, and also inWeber et al., 2005, Bioorg. Med. Chem. 13:3811-3820; Weber et al., 2006,Biorg Med Chem 14:2450-2461, and U.S. Pat. Publ. 2001-0051184 A1,published Dec. 13, 2001 (Heng).

Curcumin derivatives that exhibit improved pharmacokinetic propertiesand/or reduced toxicity are preferred. For example, curcumin derivativesthat include heteroaryl groups and/or unsaturated spacers are expectedto impart improved pharmacokinetic properties and/or reduced toxicity tothe compounds, because they are expected to be less chemically reactivein vivo. One example of preferred curcumin derivatives includes thoseinclude one or two carbonyl groups in the spacer region, including thosederivatives that preserve the enone functionality of curcumin.Derivatives that include heteroaryl groups and/or unsaturated spacersare expected to be less likely to be degraded and/or form toxic adductsor intermediates under physiological conditions. Additional curcuminderivatives not encompassed by the general definition provided above mayalso be found in the examples and schemes provided herein.

Curcumin derivatives of the invention are generally encompassed byFormula I:Ar¹—L—Ar²  (I)wherein Ar² is optional; L is a divalent linking group comprising analkylene or an alkenylene that includes between 3 and 7 backbone carbonatoms, wherein one or more of the backbone carbon atoms include acarbonyl or hydroxyl moiety; and Ar¹ and Ar² (if Ar² is present) areindependently aryl groups. Ar¹ and Ar² (if Ar² is present) may beunsubstituted or may optionally include one or more substituentsselected from the group consisting of hydroxyl, alkyl, alkenyl,haloalkyl, alkoxy, and NR₂, where R is hydrogen or alkyl. If Ar² isabsent, it may be replaced by a substituent R¹¹, including hydrogen (H).R¹¹ can be, for example, a heterocyclic group or an alkyl group,preferably an alkyl group having four or fewer carbon atoms, e.g., amethyl group. R¹¹ can alternately be an amine, a hydroxyl, or ahydrogen.Aryl Groups

Curcumin derivatives of the invention include aryl group Ar¹, which ispositioned at an end of the linker L. Curcumin derivatives of theinvention may optionally include a second aryl group Ar² that isindependently selected from Ar¹, which is positioned at the other end ofthe linker L relative to Ar¹ when present.

Preferred aryl groups include phenyl groups, naphthyl groups, thienylgroups, and pyridinium groups.

Aryl groups Ar¹ and Ar² may be substituted or unsubstituted. Preferably,substituents are selected from the group consisting of hydroxyl,halogen, alkyl, alkenyl, haloalkyl, alkoxy, amine, carboxyl, and estersubstituents.

For example, in one embodiment of the invention, Ar¹ can be a phenylgroup according to Formula II:

and Ar² can be a phenyl group according to Formula III:

The ring positions may, independently, be unsubstituted (i.e.,R=hydrogen) or one or more R groups may be substituents independentlyselected from a variety of substituents, including hydroxyl, halogen,alkyl, alkenyl, haloalkyl, alkoxy, amine, carboxyl, and estersubstituents. In further embodiments, R¹-R¹⁰ are each independentlyselected from the group including hydrogen (—H), hydroxyl (—OH), methyl(—CH₃), methoxyl (—OCH₃), dimethylamine (—N(CH₃)₂), chloro (—Cl), fluoro(—F), trifluoromethyl (—CF₃), acetoxyl, (—O(CO)CH₃) and carboxymethyl(—C(CO)OCH₃) moieties.

Divalent Linking Groups

The linker L is a spacer that preferably includes 3, 4, 5, 6 or 7 carbonatoms that form a linear carbon chain connecting the first and secondaryl groups. The carbons atoms in the carbon chain that trace outshortest path between the first and optional second aryl groups arereferred to herein as the “backbone” carbon atoms. The number ofbackbone carbon atoms is readily determined in straight chain alkylgroups. In spacers that include a cyclic alkyl group as a constituent ofthe linear chain (e.g., 38a), the backbone carbon atoms include theleast number of ring carbons possible, e.g., 3 ring carbons in 38a. Thenumber of backbone carbon atoms is used herein as a shorthand way todesignate the length of the linker being used. For example, a 7-carbonspacer is a divalent spacer that includes 7 backbone carbon atoms.Preferred embodiments of the invention include curcumin derivativeshaving an odd number of carbon atoms; e.g., 3, 5, and 7-carbon linkinggroups.

Preferably at least one of the backbone carbon atoms is included in acarbonyl (C═O) moiety. The spacer may be substituted or unsubstituted.The spacer may further be saturated or unsaturated. In a preferredembodiment, the spacer contains an odd number of carbon atoms (i.e., 3,5, or 7 carbon atoms), and at least one unsaturated carbon-carbon bond.In additional embodiments, the spacer may include a hydroxyl moiety inplace of, or in addition to, the at least one carbonyl moiety.

Curcumin derivatives of the invention include a linking group L that ispreferably covalently attached at one end to aryl group Ar¹. Optionally,the linking group L may also be covalently attached at the other end toa second aryl group, Ar², which is selected independently from Ar¹. Thelinking group L is a divalent linking group that preferably includes analkylene or an alkenylene group having between 3 and 7 backbone carbonatoms and preferably at least one carbonyl moiety. The linking group maybe substituted or unsubstituted, and may be saturated or unsaturated.Preferably, an unsaturated linking group includes conjugated doublebonds. Preferably the linking group also contains an odd number ofcarbon atoms (i.e., 3, 5, or 7 carbon atoms), and at least oneunsaturated carbon-carbon bond. In additional embodiments, the linkinggroup may include a hydroxyl moiety in place of, or in addition to, theat least one carbonyl moiety. Table 1 shows compounds with 7-carbonlinkers; Table 2 shows compounds with 5-carbon linkers; and Table 3shows compounds with 3-carbon linkers.

A divalent linking group includes two carbons with unfilled valenciesthat provide valence points where a covalent bond can be formed to anadjacent alkyl or aryl group that also includes a carbon with anunfilled valency. Generally, a valence point is represented in achemical formula by a bond that is shown as not being attached toanother group (e.g., CH₃—, wherein — represents the valence point). Inembodiments wherein the curcumin derivative lacks the second aryl groupAr², the distal valence point on the linking group can be filled withany substituent of interest, preferably a short chain alkyl group or ahydrogen (H). Compounds lacking a second aryl group may be representedby Formula IV:Ar¹—L—R¹¹  (IV)R¹¹ in Formula IV can be, for example, a heterocyclic group or an alkylgroup, preferably an alkyl group having four or fewer carbon atoms,e.g., a methyl group. R¹¹ can alternately be an amine, a hydroxyl, or ahydrogen.Curcumin Derivatives Including 7-carbon Linking Groups

In one embodiment of the invention, the curcumin derivatives include oneor two aryl groups (Ar¹ and optionally Ar²) and a linking group L thatis a 7-carbon linking group (i.e., a linking group that includes 7backbone carbon atoms). Preferably, the 7-carbon linking group includesat least one unsaturated carbon-carbon bond. Examples of 7-carbonlinking groups include—CH═CH—(CO)—CR═C(OH)—CH═CH—,—CH═CH—(CO)—CR₂—(CO)—CH═CH—, and—CH═CH—(CO)—CH═C(OH)—CH═CH—.

where R includes substituent alkyl or aryl groups comprising 10 carbonatoms or less. In some embodiments, R may be a methyl, ethyl, or benzylgroup. These linking groups are the divalent forms of 4-alkyl-1,6heptadiene-3,5-dione; 4,4-dialkyl-1,6 heptadiene-3,5-dione; andheptane-3,5-dione.

Examples of 7-C Linkers

Table 1 shows a number of examples of curcumin derivatives that includea seven carbon linker. The compounds shown contain two aryl ringsseparated by a seven carbon spacer having two carbonyls (or theequivalent keto-enol tautomer). In many, but not all, of the compounds,the spacer is unsaturated.

TABLE 1 7-Carbon Linker Analogs. 3a

3b

3c

3d

3e

3f

3g

3h

3i

6a

6b

9a

9b

11b

12b

13a

13b

14a

14b

15a

15b

16a

17b

Curcumin Derivatives Including 5-carbon Linking Groups

In a further embodiment of the invention, the curcumin derivativesinclude one or two aryl groups (Ar¹ and optionally Ar²) that are linkedby a linking group L that is a 5-carbon linking group (i.e., a linkinggroup that includes 5 backbone carbon atoms). Preferably, the 5-carbonlinking group includes at least one unsaturated carbon-carbon bond.Examples of 5-carbon linking groups include:

These linking groups are the divalent forms of 1,4-pentadiene-3-one;pentan-3-one; pentan-3-ol, 2,6; bis(methylene)cyclohexanone; and1,2,4,5-diepoxy pentan-3-one. As noted herein, curcumin derivatives mayinclude a cyclic linking group. For example, compound 31(1-methyl-2,6-diphenyl-4-piperidone), provided in Example 4 herein,provides a compound with a 5-carbon linking group that is bridged by atertiary amine to form a cyclic alkylene linking group including theheteroatom nitrogen.

Examples of 5-C Linkers

Table 2 shows a number of examples of curcumin derivatives that includea five carbon linker. The compounds shown contain two aryl ringsseparated by a five carbon spacer having a single carbonyl or hydroxyl.In many, but not all, of the compounds, the spacer is unsaturated.

TABLE 2 5-Carbon Linker Analogs. 20a

20b

20c

20d

20e

20f

20g

20i

20k

20l

20m

20n

20o

20p

20q

20r

20s

20t

20u

20v

20w

20x

20y

20z

20aa

20ab

20ac

20ae

20af

20ag

20ah

23

25

29

31

34

36a

36e

38a

38b

39b

40b

42b

43b

Curcumin Derivatives Including 3-carbon Linking Groups

In a further embodiment of the invention, the curcumin derivativesinclude one or two aryl groups (Ar¹ and optionally Ar²) that are linkedby a linking group L that is a 3-carbon linking group (i.e., a linkinggroup that includes 3 backbone carbon atoms). Preferably, the 3-carbonlinking group includes at least one unsaturated carbon-carbon bond. Anexample of a 3-carbon linking group is —CH═CH—CH(O)—; i.e., a divalentform of propenone.

Examples of 3-C Linkers

Table 3 shows a number of examples of curcumin derivatives that includea three carbon linker. The compounds shown generally have an unsaturatedthree-carbon spacer having a single carbonyl. While most of the examplesshown have two aryl groups separated by the spacer, several of theembodiments include only a single aryl group. In the examples thatinclude only a single aryl group, a methyl group is provided at theother end of the linking group. Compound 52b includes the heteroatom Nin place of one of the backbone carbon atoms; however, this is stillconsidered a 3-C linker in that 3 atoms (C, N, and C) are present alongthe shortest bridge between the two aryl groups.

TABLE 3 3-Carbon Linker Analogs. 35a

35e

35q

45a

45b

46a

46ad

46ak

46al

48a

48ad

50b

52b

Additional Curcumin Derivatives

Curcumin derivatives of the invention may include a variety of linkinggroups and Ar groups while retaining biological activity, so long asthey provide a structure that will inhibit NK-κB, AP-1 and/or GSTP1-1activity. Accordingly, additional curcumin analogs are contemplated.Since analogs that contain a central methylene substituent on the7-carbon spacer have shown significant activity, analogs containing acentral group other than methyl or benzyl may also exhibit significantinhibition of NF-κB, AP-1 and/or GSTP1-1 activation. These includecurcumin analogs containing central methylene substituents such asethyl, propyl, butyl, isopropyl and substituted benzyl groups as shownbelow:

These compounds can be synthesized using the procedures shown in Schemes1 and Scheme 2. The descriptions and details for these procedures arethe same as those described for Schemes 8 and 9.

Additional analogs that are contemplated are those having a pyridinering with and without a central methylene substituent on the 7-carbonspacer such as those shown below. Analogs without a central methylenesubstituent can be prepared according to Pabon's method shown in Scheme3. The descriptions and details for this procedure are the same asdescribed for Scheme 6. The analogs having a pyridine aryl ring with acentral methylene substituent on the 7-carbon spacer can be synthesizedusing a procedure described in Scheme 1 using 2, 3 or 4-pyridinecarboxaldehyde.

Many curcumin analogs which have a 5-carbon spacer possess significantactivity. Additional active analogs in this series may containsubstituents such as hydroxy and methoxy groups on the aryl rings.Therefore, other substituents and their positions on the aryl rings mayalso provide significant inhibition of NF-κB, AP-1 and/or GSTP1-1activation. Examples of these analogs are shown below:

These new analogs can be prepared as shown in Scheme 4. The descriptionsand details for this procedure are the same as described for Scheme 13.

Although analogs having 3-carbon spacers were generally not as active asanalogs having 7-carbon or 5-carbon spacers, additional analogs mayprovide significant inhibition of NF-κB, AP-1 and/or GSTP1-1 activation.Relatively few analogs in this series containing a heterocyclic ring onthe spacer have been synthesized. Analogs having different substituentson the aryl rings may provide significant inhibition of NF-κB, AP-1,and/or GSTP1-1 activation. In addition, analogs that contain differentsubstituents on the nitrogen of the heterocyclic ring may providesignificant inhibition of NF-κB, AP-1, and/or GSTP1-1 activation.Examples of these series 3 analogs are shown below:

These analogs can be synthesized as shown in Scheme 5. The descriptionsand details for this procedure are the same as those described forScheme 36.

Additional curcumin derivatives of the invention that are notencompassed by the embodiments provided above may also be found in theexamples and schemes provided herein.Disease Treatment Using Curcumin Derivatives

Treatment, as defined herein, is the amelioration of the symptomsassociated with disease. Symptoms may be reduced either by decreasingthe level of the disease itself, or by decreasing the symptomsassociated with the disease. The subject of the treatment is preferablya mammal, such as a domesticated farm animal (e.g., cow, horse, pig) orpet (e.g., dog, cat). More preferably, the subject is a human.

As noted herein, and without being bound by any particular theory, onemechanism by which administration of curcumin derivatives may treatdisease is through inhibition of the activity of AP-1, NF-κB and/orGSTP1-1. Inhibition of NF-κB results in a decrease in NF-κB activity,and includes direct inhibition and indirect inhibition. Directinhibition is the direct effect of a curcumin derivative on NF-κB andits activity. For example, one type of direct inhibition of NF-κB is ablock of NF-κB DNA interactions. Indirect inhibition, on the other hand,involves the effect of a curcumin derivative on other compounds involvedin the regulation of NF-κB that leads to a decrease in NF-κB activity.For example, as phosphorylation of the NF-κB regulator IκB by IκBkinases (IKK) or Src family kinases (SFK) results in a dysregulation ofNF-κB, and an according increase in NF-κB activity, inhibition of IKK orSFK by curcumin derivatives provides an example of indirect inhibition.

Inhibition of AP-1 results in a decrease in AP-1 activity, and includesdirect inhibition and indirect inhibition. Direct inhibition is thedirect effect of a curcumin derivative on AP-1 (or its subunits) and itsactivity. Indirect inhibition, on the other hand, involves the effect ofa curcumin derivative on other compounds involved in the regulation ofAP-1 that leads to a decrease in AP-1 activity. For example, indirectinhibition of AP-1 activity may occur as a result of an affect on AP-1activating proteins such as mitogen-activated protein kinases (MAPK) orc-Fos-regulating kinase (FRK).

Inhibition of GSTP1-1 results in a decrease GSTP1-1 activity, andincludes direct inhibition and indirect inhibition. Direct inhibition isthe direct effect of a curcumin derivative on GSTP1-1 (or its subunits)and its activity. Indirect inhibition, on the other hand, involves theeffect of a curcumin derivative on other compounds involved in theregulation of GSTP1-1 that leads to a decrease in GSTP1-1 activity.Various methods for inhibiting of GSTP1-1 are exemplified in Examples 12and 13.

Alzheimer's Disease

In one aspect, the present invention provides a method of using curcuminderivatives to treat a subject with Alzheimer's disease. The presentinvention also provides a method of using curcumin derivatives to treatsymptoms of Alzheimer's disease in a subject with Alzheimer's disease.Curcumin derivatives treat Alzheimer's disease through one or morebiochemical mechanisms. For example, without being bound by theory,administration of curcumin derivatives may treat Alzheimer's disease byinhibiting the activity of AP-1 and/or NF-κB. Decreasing the activity ofAP-1 and/or NF-κB may, in turn, lead to a decrease in inflammation.

As another example, again without being bound by theory, administrationof curcumin derivatives may treat Alzheimer's disease through an effecton the Aβ peptide, for example by inhibiting the formation of Aβoligomers and fibrils, reducing Aβ peptide aggregation, or by reducingthe Aβ amyloid burden of subjects with Alzheimer's disease. The effectsof curcumin derivatives on Aβ peptide aggregation may include binding toAβ peptide aggregates and/or effects on Aβ peptide conformation. Effectson Aβ peptide conformation include destabilization of the β-sheetconformation of Aβ peptide aggregates, and/or the stabilization ofnon-aggregated Aβ peptide α-helical/random coil conformation. Theeffects of curcumin derivatives may further include a decrease in thecytotoxicity of Aβ peptide aggregates, or a decrease in glial cellactivation by Aβ peptide aggregates.

Symptoms of Alzheimer's disease include, for example, formation ofamyloid plaques and neurofibrillary tangles, chronic brain inflammation,glial cell activation, and cognitive decline. A number of other symptomsare known and can be readily identified by one skilled in the art.

Type 2 Diabetes

In another aspect, the present invention provides a method of usingcurcumin derivatives to treat a subject with type 2 diabetes. Thepresent invention also provides a method of using curcumin derivativesto treat symptoms of diabetes in a subject with type 2 diabetes such asinflammation or insulin resistance.

Symptoms of type 2 diabetes include, for example, insulin resistance andinflammation. A number of other symptoms are known and can be readilyidentified by one skilled in the art.

Other Inflammatory Diseases or Conditions

In should be understood that the present invention provides a method ofusing curcumin derivatives to treat a subject with any disease orcondition characterized by inflammation, including Alzheimer's disease,diabetes (particularly type 2 diabetes), cancer, cystic fibrosis,rheumatoid arthritis, asthma, inflammatory bowel disease, ulcerativecolitis, atherosclerosis and stroke.

Assistive or Adjuvant Treatment for Cancer

In another aspect, the present invention pertains generally to treatmentof cancer or a precancerous condition such as dysplasia or hyperplasia,by administering a curcumin derivative of the invention. Administrationof the curcumin derivative may advantageously inhibit the activityGlutathione S-transferase P1-1 (GSTP1-1), NFκB and/or AP-1. Inhibitionof GSTP1-1 may occur by affecting gene transcription and/or by directeffects on enzyme activity.

In a preferred embodiment, administration of the curcumin derivative iseffected in combination with the administration of anotherchemotherapeutic agent. The curcumin derivative can be administeredbefore, during of after the administration of the chemotherapeuticagent. Administration of the curcumin derivative is especiallyadvantageous in cases where the cancer cells may develop or havedeveloped resistance to the chemotherapeutic agent; and/or when thecancer cells overexpress GSTP1-1. Expression of GSTP1-1 may allow thecancer cell to pump out the chemotherapeutic agent, and by inhibitingthe activity of GSTP1-1, the curcumin derivative may preserve or prolongthe cytostatic or cytotoxic effects of the chemotherapeutic agent. Thepresent invention is particularly useful for improving the effectivenessof chemotherapeutic agents by preventing GSTP1-1's inhibition ofpro-apoptotic factors, particularly c-Jun N-terminal kinase (JNK).

Curcumin was shown recently to inhibit apoptosis in cancer cells in partthrough its ability to inhibit the expression of GSTP1-1 mRNA andprotein, which was demonstrated to be the result of inhibition of theactivation of NFκB. This observation that compounds such as curcumin canblock activation of NFκB raises the possibility that synthetic drugs canbe developed that are more potent than curcumin, and that these drugswill promote apoptosis in cancer cells. These drugs could sensitizecancer cells to conventional adjuvant chemotherapy by blocking theNFκB-dependent development of the anti-apoptotic pro-survival state, andinhibit the expression of GSTP1-1. In addition, curcumin inhibits theGSTP1-1 catalyzed conjugation of glutathione with electrophiles.Furthermore, curcumin inhibits the proliferation of a variety of tumorcells and has anti-metastatic activity, possibly owing to its ability toinduce apoptosis by inhibiting NFκB.

Curcumin contains two alpha, beta-unsaturated carbonyl groups, one ofwhich exists as the enol tautomer. Curcumin reacts with glutathione;this reaction is accelerated by GSTP1-1, indicating that curcumin is asubstrate of GSTP1-1, albeit a poor substrate. Curcumin also inhibitsGSTP1-1 in its conjugation of glutathione with other electrophiles,suggesting that curcumin is both a substrate and an inhibitor ofGSTP1-1. This is consistent with the known inhibition of GSTP1-1 by theflavonoid quercetin, which, like curcumin, is also a polyphenol.Curcumin itself has low bioavailability and therefore is not a promisingdrug.

In view of the reports that suggest a role for curcumin in cancertherapy as a direct inhibitor of GSTP1-1, as well as a down-regulator ofGSTP1-1 through inhibition of NFκB, analogues of curcumin with goodbioavailability can be developed as new anti-cancer drugs that mayinhibit the catalytic activity of GSTP1-1, thereby sensitizing cancercells to conventional chemotherapy by drugs that normally aremetabolized through GSTP1-1 catalyzed conjugation with glutathione;and/or, through inhibition of GSTP1-1 and/or NFκB the curcuminderivatives, contribute to improved chemotherapeutic drug sensitivity ofcancer cells by promoting the pro-apoptotic state. These analogs thusmay have a dual mechanism of action—both the inhibition of the catalyticactivity of GSTP1-1 and the down-regulation of GSTP1-1 transcriptionthrough inhibition of NFκB. GSTP1-1 inhibitors may limit the ability ofGSTP1-1 to inactivate other cancer drugs and may prove to be synergisticwhen combined with important chemotherapeutic drugs including platinums,taxanes and anthracyclines. The invention is not limited to the types ofcancer that can be treated. Cancers that can be treated include breastcancer, ovarian cancer, prostate cancer, non-Hodgkin's lymphoma, andleukemia.

Identification of Agents

Another aspect of the invention includes methods for identifyingtherapeutic curcumin derivatives that may be used to treat a subjectafflicted with a disease. Potential agents suitable for testing arereferred to herein as “candidate” agents. In one embodiment, the methodinvolves exposing AP-1, NF-κB or GSTP1-1 to the candidate agent anddetermining whether or not its activation by an AP-1, NF-κB or GSTP1-1activator is inhibited. As AP-1 and NF-κB are transcription factors,their activation is most readily evaluated in a cell assay. However,AP-1 or NF-κB activation can also be evaluated in cell-free systemsusing techniques readily known by those skilled in the art. Sources forcandidate agents include, for instance, chemical compound libraries, andextracts of plants and other vegetations.

For example, in one embodiment, the method for identifying a therapeuticcurcumin derivative involves contacting a cell containing NF-κB with acandidate curcumin derivative, contacting the cell with an NF-κBactivator (e.g., TNF-α or IL-1) and determining the effect on NF-κBactivation by the curcumin derivative. Preferably, the cell contains“activatable” NF-κB; that is, the cell has or retains the capacity forNF-κB activation. A candidate agent that results in a decrease of NF-κBactivation is accordingly identified by this method as a therapeuticcurcumin derivative. Cells that may be used to test for a decrease ofNF-κB activation include, for example, adipose and endothelial cells.For example, a cell assay suitable for identifying curcumin derivativesthat are useful for treating a subject with type 2 diabetes is providedby Example 3, herein.

In a further exemplary embodiment, the method for identifying atherapeutic curcumin derivative involves contacting a cell containingAP-1 with a candidate curcumin derivative, contacting the cell with anAP-1 activator (e.g., TNF-α or phorbol 12-myristate 13-acetate) anddetermining the extent of the effect on AP-1 activation by the curcuminderivative. Preferably, the cell contains “activatable” AP-1; that is,the cell has or retains the capacity for AP-1 activation. A candidateagent that results in a decrease of AP-1 activation is accordinglyidentified by this method as a therapeutic curcumin derivative. Cellsthat may be used to test for a decrease of AP-1 activation include, forexample, adipose and endothelial cells. For example, a cell assaysuitable for identifying curcumin derivatives that are useful fortreating a subject with type 2 diabetes is provided by Example 5,herein.

In another exemplary embodiment, the method for identifying atherapeutic curcumin derivative involves contacting a cell containingGSTP1-1 with a candidate curcumin derivative, contacting the cell with aGSTP1-1 activator (e.g., TNF-α) and determining the extent of the effecton GSTP1-1 activation by the curcumin derivative. Preferably, the cellcontains “activatable” GSTP1-1; that is, the cell has or retains thecapacity for GSTP1-1 activation. A candidate agent that results in adecrease of AP-1 activation is accordingly identified by this method asa therapeutic curcumin derivative. A cell assay suitable for identifyingcurcumin derivatives that are useful for assistive or adjuvantchemotherapeutic treatment, for example, is provided by Examples 12 and13.

As another example, therapeutic curcumin derivatives can be identifiedby observing the effect of curcumin derivatives on cell activation byinflammation activators. Inflammation activators, as defined herein,include compounds that stimulate cells to result in an increase ininflammation. Activated cells may, for example, release higher levels ofcytokines (e.g., IL-1 or IL-6) than unactivated cells. Examples ofinflammation activators include AP-1 and NF-κB. As AP-1 and NF-κB aretranscription factors, their activation is most readily evaluated in acell assay. However, AP-1 or NF-κB activation can also be evaluated incell-free systems using techniques readily known by those skilled in theart.

A therapeutic curcumin derivative that is useful as an anti-Alzheimer'sagent can be identified by evaluating the effect of the candidate agenton symptoms or biochemicals associated with Alzheimer's diseaseneuropathology. For example, in one embodiment, a therapeutic curcuminderivative can be identified by contacting a brain cell that includes aninflammation activator with a curcumin derivative and determining theeffect of the curcumin derivative on brain cell activation by theinflammation activator. A candidate agent that reduces brain cellactivation is accordingly identified by this method as a curcuminderivative that may be used to treat a subject with Alzheimer's disease.Brain cells, as defined herein, include neurons and glial cells. Glialcells further include microglia, which are a specialized form ofmacrophage cells. Cell activation may be evaluated, for example, bydetermining the level of cytokines (e.g., IL-1 or IL-6) released orexpressed by the cells.

Curcumin derivatives effective to treat Alzheimer's disease may beidentified by directly determining their effect on the activity of NF-κBor AP-1, as detailed elsewhere herein. Evaluation of the effect on NF-κBor AP-1 can be carried out in a variety of different types of cells. Forexample, a cell assay involving activation of NF-κB that is suitable foridentifying curcumin derivatives that are useful for treating a subjectwith Alzheimer's disease is provided by Example 3. An example of a cellassay involving activation of AP-1 that is suitable for identifyingcurcumin derivatives that are useful for treating Alzheimer's disease isprovided by Example 5. Therapeutic curcumin derivatives may also beidentified using high-throughput screening (HTS), as described inExample 9.

Another method for identifying therapeutic curcumin derivativeseffective to treat Alzheimer's disease includes observing the effects ofcurcumin derivatives on the Aβ peptide. These effects may includeinhibition of the formation of Aβ oligomers and fibrils, a decrease inAβ peptide aggregation, or a decrease in the Aβ amyloid burden ofsubjects with Alzheimer's disease. More specifically, the effects ofcurcumin derivatives on Aβ peptide aggregation may include binding to Aβpeptide aggregates and/or effects on Aβ peptide conformation. Effects onAβ peptide conformation may include destabilization of the β-sheetconformation of Aβ peptide aggregates, and/or the stabilization ofnon-aggregated Aβ peptide α-helical/random coil conformation. Theeffects of curcumin derivatives may further include a decrease in thecytotoxicity of Aβ peptide aggregates, or a decrease in glial cellactivation by Aβ peptide aggregates. These effects may be determinedusing a number of different assays, such as those described in Example10.

For example, one method for identifying antiAlzheimer curcuminderivatives includes contacting a solution that includes an Aβ peptidewith a curcumin derivative and determining the effect of the curcuminderivative on aggregation by the Aβ peptide. A curcumin derivative thatreduces aggregation of the Aβ peptide is thus identified as anantiAlzheimer curcumin derivative. The Aβ peptide may includederivatives of the Aβ peptide such as N-terminal biotinylated A(1-40)peptide. The effect of the curcumin derivative on aggregation by the Aβpeptide can be determined using a variety of different techniques knownto those skilled in the art, such as use of an immunological assay todetermine the amount of aggregated Aβ peptide formed.

Candidate agents may also be tested in animal models. In one embodiment,the animal model is selected for the study of diabetes. The study ofdiabetes in animal models (for instance, mice) is a commonly acceptedpractice by those skilled in the art. Examples of mouse models for type2 diabetes include C57BLKs, CBA/CaJ, NON/LtJ, andB6.HRS(BKS)-Cpe^(fat)/J mouse strains. Further strains may be obtainedfrom JAX Mice Diabetes & Obesity Research Models, The Jackson Laboratory(Bar Harbour, Me.). Results are typically compared between controlanimals treated with candidate agents and the control littermates thatdid not receive treatment. Transgenic animal models are also availableand are commonly accepted as models for human disease (see, forinstance, Greenberg et al., Proc. Natl. Acad. Sci. USA, 92:3439-3443(1995)). Candidate agents can be used in these animal models todetermine if a candidate agent decreases one or more of the symptomsassociated with the diabetes such as inflammation or insulin resistance.

In another embodiment, the animal model is one developed by thoseskilled in the art for use in studies of Alzheimer's disease. Forexample, rat and mouse models of Alzheimer's disease have been developedthat overexpress the Aβ peptide. See, for example, the review of geneticmouse models of Alzheimer's disease by Mineur et al., Neural Plast.(2005) 12(4), 299-310. Results are typically compared between controlanimals treated with candidate agents and the control littermates thatdid not receive treatment. Transgenic animal models are also availableand are commonly accepted as models for human disease (see, forinstance, Greenberg et al., Proc. Natl. Acad. Sci. USA, 92:3439-3443(1995)). Candidate agents can be used in these animal models todetermine if a candidate agent decreases one or more of the symptomsassociated with Alzheimer's disease, such as inflammation or Aβ peptideaggregation.

Administration and Formulation of Curcumin Derivatives

The present invention provides a method for using a composition thatincludes one or more small molecule inhibitors of the invention to treata subject afflicted with a disease by administering curcumin derivativesalone, or along with one or more pharmaceutically acceptable carriers.One or more curcumin derivatives with demonstrated biological activitycan be administered to a subject in an amount alone or together withother active agents and with a pharmaceutically acceptable buffer. The acomposition that includes one or more small molecule inhibitors of theinvention can be combined with a variety of physiological acceptablecarriers for delivery to a patient including a variety of diluents orexcipients known to those of ordinary skill in the art. For example, forparenteral administration, isotonic saline is preferred. For topicaladministration, a cream, including a carrier such as dimethylsulfoxide(DMSO), or other agents typically found in topical creams that do notblock or inhibit activity of the peptide, can be used. Other suitablecarriers include, but are not limited to, alcohol, phosphate bufferedsaline, and other balanced salt solutions.

Methods of administering small molecule therapeutic agents arewell-known in the art. Reference is made, for example, to U.S. Pat.Publ. 2001-0051184 A1, published Dec. 13, 2001 (Heng) concerningillustrative modes of administration of curcumin analogs as well asdosage amounts and protocols.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the active agentinto association with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active agent into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations. Themethods of the invention include administering to a subject, preferablya mammal, and more preferably a human, the composition of the inventionin an amount effective to produce the desired effect. The curcuminderivatives can be administered as a single dose or in multiple doses.Useful dosages of the active agents can be determined by comparing theirin vitro activity and the in vivo activity in animal models. Methods forextrapolation of effective dosages in mice, and other animals, to humansare known in the art; for example, see U.S. Pat. No. 4,938,949.

The agents of the present invention are preferably formulated inpharmaceutical compositions and then, in accordance with the methods ofthe invention, administered to a subject, such as a human patient, in avariety of forms adapted to the chosen route of administration. Theformulations include, but are not limited to, those suitable for oral,rectal, vaginal, topical, nasal, ophthalmic, or parental (includingsubcutaneous, intramuscular, intraperitoneal, intratumoral, andintravenous) administration.

Formulations suitable for parenteral administration conveniently includea sterile aqueous preparation of the active agent, or dispersions ofsterile powders of the active agent, which are preferably isotonic withthe blood of the recipient. Parenteral administration of curcuminderivatives (e.g., through an I.V. drip) is an additional form ofadministration. Isotonic agents that can be included in the liquidpreparation include sugars, buffers, and sodium chloride. Solutions ofthe active agent can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions of the active agent can be prepared inwater, ethanol, a polyol (such as glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, glycerol esters,and mixtures thereof. The ultimate dosage form is sterile, fluid, andstable under the conditions of manufacture and storage. The necessaryfluidity can be achieved, for example, by using liposomes, by employingthe appropriate particle size in the case of dispersions, or by usingsurfactants. Sterilization of a liquid preparation can be achieved byany convenient method that preserves the bioactivity of the activeagent, preferably by filter sterilization. Preferred methods forpreparing powders include vacuum drying and freeze drying of the sterileinjectible solutions. Subsequent microbial contamination can beprevented using various antimicrobial agents, for example,antibacterial, antiviral and antifungal agents including parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Absorptionof the active agents over a prolonged period can be achieved byincluding agents for delaying, for example, aluminum monostearate andgelatin.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as tablets, troches, capsules,lozenges, wafers, or cachets, each containing a predetermined amount ofthe active agent as a powder or granules, as liposomes containing thecurcumin derivatives, or as a solution or suspension in an aqueousliquor or non-aqueous liquid such as a syrup, an elixir, an emulsion, ora draught. Such compositions and preparations typically contain at leastabout 0.1 wt-% of the active agent. The amount of curcumin derivatives(i.e., active agent) is such that the dosage level will be effective toproduce the desired result in the patient.

Nasal spray formulations include purified aqueous solutions of theactive agent with preservative agents and isotonic agents. Suchformulations are preferably adjusted to a pH and isotonic statecompatible with the nasal mucous membranes. Formulations for rectal orvaginal administration may be presented as a suppository with a suitablecarrier such as cocoa butter, or hydrogenated fats or hydrogenated fattycarboxylic acids. Ophthalmic formulations are prepared by a similarmethod to the nasal spray, except that the pH and isotonic factors arepreferably adjusted to match that of the eye. Topical formulationsinclude the active agent dissolved or suspended in one or more mediasuch as mineral oil, petroleum, polyhydroxy alcohols, or other basesused for topical pharmaceutical formulations.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: a binder such as gum tragacanth, acacia, cornstarch or gelatin; an excipient such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose, fructose, lactose, or aspartame; and a natural orartificial flavoring agent. When the unit dosage form is a capsule, itmay further contain a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials may be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules may be coated with gelatin,wax, shellac, sugar, and the like. A syrup or elixir may contain one ormore of a sweetening agent, a preservative such as methyl- orpropylparaben, an agent to retard crystallization of the sugar, an agentto increase the solubility of any other ingredient, such as a polyhydricalcohol, for example glycerol or sorbitol, a dye, and flavoring agent.The material used in preparing any unit dosage form is substantiallynontoxic in the amounts employed. The active agent may be incorporatedinto sustained-release preparations and devices.

The curcumin derivatives of the invention can be incorporated directlyinto the food of the mammal's diet, as an additive, supplement, or thelike. Thus, the invention further provides a food product containing acurcumin derivative of the invention. Any food is suitable for thispurpose, although processed foods already in use as sources ofnutritional supplementation or fortification, such as breads, cereals,milk, and the like, may be more convenient to use for this purpose.

Small molecule inhibitors such as curcumin derivatives are well-suitedfor inhibiting AP-1 or NF-κB activity, as they are usually easilysynthesized and readily taken up by mammalian cells. In someembodiments, the small molecule inhibitor is derivatized or conjugatedwith a carrier molecule according to methods well known in the art, soas to increase targeting efficiency and/or the rate of cellular uptake,for example by being covalently linked to a ligand that binds to a cellsurface receptor.

Preparation of Curcumin Derivatives

Compounds of the invention may be synthesized by synthetic routes thatinclude processes analogous to those well known in the chemical arts,particularly in light of the description contained herein. The startingmaterials are generally available from commercial sources such asAldrich Chemicals (Milwaukee, Wis., USA) or are readily prepared usingmethods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-19, Wiley, New York, (1967-1999 ed.); AlanR. Katritsky, Otto Meth-Cohn, Charles W. Rees, Comprehensive OrganicFunctional Group Transformations, v 1-6, Pergamon Press, Oxford,England, (1995); Barry M. Trost and Ian Fleming, Comprehensive OrganicSynthesis, v. 1-8, Pergamon Press, Oxford, England, (1991); orBeilsteins Handbuch der organischen Chemie, 4, Aufl. Ed.Springer-Verlag, Berlin, Germany, including supplements (also availablevia the Beilstein online database)).

For illustrative purposes, the reaction schemes depicted below providepotential routes for synthesizing the compounds of the present inventionas well as key intermediates. For more detailed description of theindividual reaction steps, see the EXAMPLES section below. Generally,compounds of the present invention are prepared by reacting a pair ofaryl aldehydes using an aldol reaction. For example, curcuminderivatives including a 7-carbon linker may be prepared by reacting2,4-pentanedione with a substituted arylaldehyde in an aldol-typereaction according to the procedure described by Pabon (Pabon, H. J. J.Recueil 83, 379 (1964)). In a further example, curcumin derivativesincluding a 5-carbon linker may be prepared by reaction of acetone withsubstituted arylaldehydes in a base catalyzed aldol reaction, asdescribed by Masuda et al. (Masuda et al., Phytochemistry 32, 1557(1993)), and curcumin derivatives including a 3-carbon linker (alsoreferred to as chalcones) can be prepared by reaction of a substitutedarylaldehyde with a substituted aceto-aryl compound (e.g., acetophenone)in a base catalyzed aldol reaction as described by Kohler and Chadwell(Kohler, E. P.; Chadwell, H. M. Org. Synth., Coll. 1, 78 (1932)).

Those skilled in the art will appreciate that other synthetic routes maybe used to synthesize the compounds of the invention. Although specificstarting materials and reagents are depicted in the reaction schemes anddiscussed below, other starting materials and reagents can be easilysubstituted to provide a variety of derivatives and/or reactionconditions. In addition, many of the compounds prepared by the methodsdescribed below can be further modified in light of this disclosureusing conventional methods well known to those skilled in the art.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scope ofthe invention as set forth herein.

EXAMPLES Example 1 Chemical Synthesis of Curcumin Derivatives

Several derivatives were synthesized that have some structuralsimilarity to curcumin. The following is a discussion of the analogsthat were synthesized and the methods used to accomplish the structuralchanges. Spectral data were useful in characterizing structural changesin the molecules. Proton and carbon nuclear magnetic resonancespectroscopies (NMR) were used to detect functional groups in thecurcumin analogs. The following schemes summarize the procedures used toprepare the three series of curcumin analogs. Analogs in series 1, whichretain the 7-carbon spacer contained in curcumin, were prepared as shownin Schemes 6-11. Analogs in series 2, which contain a 5-carbon spacer,were prepared as shown in Schemes 12-27. Analogs in series 3, whichcontain a 3-carbon spacer, were prepared as shown in Schemes 28-37.

Synthesis of 7-Carbon Spacer Analogs

Analogs 3a-3i, contain two aryl rings separated by an unsaturated7-carbon spacer having two carbonyls (Schemes 1 and 2). The aryl ringscontain different substituents in various positions on the ring. Theseanalogs were designed to test the importance of the type of substituentand its location on the aryl ring. Analogs 3a-3h, as shown in Scheme 6,were prepared following the procedure described by Pabon (Pabon,Recueil, 83, 379-386 (1964)). 2,4-Pentanedione (2) was reacted withboric anhydride to give the boron/pentanedione complex. The complex wasthen reacted with the appropriately substituted benzaldehyde (1a-1h),tributyl borate, and butylamine in dry ethyl acetate in an aldol typereaction followed by hydrolysis with warm dilute hydrochloric acid togive curcumin (3a) or one of its analogs 3b-3h.

The formation of the products was verified by proton NMR by theappearance of a pair of doublets in the aromatic region with J values of15.5-16.5 Hz for the alkene protons present in the spacer. Also observedin the proton NMR was the loss of a signal at ˜10 ppm for the aldehydeproton in the starting benzaldehyde and the loss of signals at 1.89 ppmand 2.08 ppm for the methyl protons on 2,4-pentanedione (2). Thestructures were also verified by carbon NMR by the appearance of asignal at ˜182 ppm for the keto-enol carbonyl carbon and the loss of asignal at ˜195 ppm for the aldehyde carbon in the starting benzaldehyde(1a-1h). Also absent from the carbon NMR were signals at 24.1 ppm and30.2 ppm for the methyl carbons on 2,4-pentanedione (2).

Analog 3d, which is not in the literature, was verified by elementalanalysis.

Scheme 7 describes the synthesis of analog 3i. Analog 3i was alsoprepared following the procedure described by Pabon (Pabon, Recueil, 83,379-386 (1964)). 2,4-Pentanedione (2) was reacted with boric anhydridein dry ethyl acetate at 40° C. to give the boron/pentanedione complex.The complex was then reacted with 3,4-dimethoxybenzaldehyde (1i),tributyl borate, and butylamine in dry ethyl acetate at 40° C. in analdol type reaction followed by hydrolysis with warm dilute hydrochloricacid to give analog 3i. The formation of the product was verified byproton NMR by the appearance of a pair of doublets in the aromaticregion with J values of 15.9 Hz for the alkene protons present in thespacer. Also observed in the proton NMR was the loss of a signal at 9.85ppm for the aldehyde proton in the starting benzaldehyde (1i) and theloss of signals at 1.89 ppm and 2.08 ppm for the methyl protons on2,4-pentanedione (2). The structure was also verified by carbon NMR bythe appearance of a signal at 183.0 ppm for the keto-enol carbonylcarbon and the loss of a signal at 190.9 ppm for the aldehyde carbon inthe starting benzaldehyde (1i). Also absent from the carbon NMR weresignals at 24.1 ppm and 30.2 ppm for the methyl carbons on2,4-pentanedione (2).

Two additional curcumin analogs, 6a and 6b, were prepared as shown inScheme 8. Analogs 6a and 6b contain two aryl rings separated by anunsaturated 7-carbon spacer having two carbonyls and a single methylsubstituent attached to the central methylene carbon. These analogs weredesigned to test the importance of a methyl substituent on the centralmethylene carbon. 3-Methyl-2,4-pentanedione (5) was first synthesized byreaction of 2,4-pentanedione (2) with potassium carbonate and methyliodide (4) in acetone at 56° C. in a substitution reaction following theprocedure described by Markham and Price (Markham et al., Org. Synth.Coll. Vol. V. 785-790). This reaction gave the monomethyl substitutedproduct as the major product along with small amounts of both unreacted2,4-pentanedione (2) and of the dimethyl substituted product. Theformation of the product was verified by proton NMR by the appearance ofa doublet at 1.12 ppm for the methyl protons and a quartet at 3.52 ppmfor the remaining methylene proton. Analogs 6a and 6b were then preparedfrom compound 5, following the procedure described by Pabon (Pabon,Recueil, 83, 379-386 (1964)), by reaction with boric anhydride under anitrogen atmosphere to give the boron/pentanedione complex. The complexwas then reacted with 4-hydroxy-3-methoxybenzaldehyde (1a) orbenzaldehyde (1b), tributyl borate, and butylamine in an aldol typereaction followed by hydrolysis with warm dilute hydrochloric acid togive 6a and 6b respectively. The formation of products was verified byproton NMR by the appearance of a pair of doublets in the aromaticregion with J values of 15.5 Hz for the alkene protons in the spacer andthe loss of signals at 1.92 ppm and 2.00 ppm for the terminal methylprotons on 3-methyl-2,4-pentanedione (5). The structures were alsoverified by carbon NMR by the appearance of a signal at ˜182.2 ppm forthe keto-enol carbonyl carbon. The carbon NMR also showed the loss ofsignals at 23.0 ppm and 28.4 ppm for the terminal methyl carbons on3-methyl-2,4-pentanedione (5). The carbon NMR of analog 6b also showedthe loss of a signal at 192.1 ppm for the aldehyde carbon in thestarting benzaldehyde (1b), whereas in analog 6a, a signal is present at196.0 ppm due to the carbonyl carbon of the diketo form and not thealdehyde carbon of the starting benzaldehyde (1b). Both analogs 6a and6b, which are not in the literature, were verified by elementalanalysis.

Two additional curcumin analogs, 9a and 9b, were prepared as shown inScheme 9. Analogs 9a and 9b contain two aryl rings separated by anunsaturated 7-carbon spacer having two carbonyls and a single benzylsubstituent attached to the central methylene carbon. These analogs weredesigned to test the importance of a benzyl substituent on the centralmethylene carbon. The starting material 3-benzylidene-2,4-pentanedione(7), was prepared by a Knoevenagel condensation reaction of2,4-pentanedione (2) with benzaldehyde (1b), glacial acetic acid andpiperdine in benzene at 65° C. following the procedure described byAntonioletti (Antonioletti et al., Tetrahedron 58(3), 589-596 (2002)).The formation of the product was verified by proton NMR by theappearance of a signal at 7.45 ppm for the alkene proton and the loss ofa signal at 5.37 ppm for the central methylene proton on compound 2.3-Benzyl-2,4-pentanedione (8) was prepared by reaction of compound 7with palladium on activated carbon under a hydrogen atmosphere on a Parrapparatus in a reduction reaction following the procedure described byVenkateswarlu (Venkateswarlu et al., Asian J. Chem. 12(1), 141-144(2000)). The formation of the product was verified by proton NMR by theappearance of triplet at 4.01 ppm for the central methylene proton and adoublet at 3.11 ppm for the benzylic protons of the diketo form ofcompound 8. Also observed in the proton NMR is a singlet at 3.62 ppm forthe benzylic protons of the keto-enol form of compound 8. The proton NMRalso shows the loss of a signal at 7.45 ppm for the alkene proton incompound 7. Analogs 9a and 9b were then prepared from compound 8,following the procedure described by Pabon (Pabon, Recueil, 83, 379-386(1964)), by reaction with boric anhydride under a nitrogen atmosphere togive the boron/pentanedione complex. The complex was then reacted with4-hydroxy-3-methoxybenzaldehyde (1a) or benzaldehyde (1b), tributylborate, and butylamine in an aldol type reaction followed by hydrolysiswith warm dilute hydrochloric acid to give 9a and 9b respectively. Theformation of the products was verified by proton NMR by the appearanceof a pair of doublets in the aromatic region with J values of 15.1-15.6Hz for the alkene protons in the spacer and the loss of signals at ˜2.05ppm for the methyl

protons of 3-benzyl-2,4-pentanedione (8). The structures were alsoverified by carbon NMR by the appearance of a signal at 183.3 ppm forthe keto-enol carbonyl carbon. Also observed in the carbon NMR was theloss of signals at 22.9 ppm and 29.4 ppm for the methyl carbons of3-benzyl-2,4-pentanedione (8). The carbon NMR of analog 9b also showedthe loss of a signal at 192.1 ppm for the aldehyde carbon in thestarting benzaldehyde (1b); whereas in analog 9a, a signal was presentat 194.0 ppm for the carbonyl carbon of the diketo form of the analog.Analog 9a, which is not in the literature, was verified by elementalanalysis.

Two additional curcumin analogs, 11b and 12b, were prepared as shown inScheme 10. Analogs 11b and 12b contain two aryl rings separated by anunsaturated 7-carbon spacer having two carbonyls. Analog 11b containstwo methyl substituents attached to the central methylene carbon,whereas analog 12b contains two benzyl substituents attached to thecentral methylene carbon. These analogs were designed to test theimportance of two substituents on the central methylene carbon. Analogs11b and 12b were prepared by reaction of analog 3b with sodiumhydroxide, tetrabutylammonium chloride and either methyl iodide (4) orbenzyl bromide (10) in dichloromethane at 40° C. in a substitutionreaction following the procedure described by Pedersen (Pedersen et al.,Liebigs Ann. Chem. 8, 1557-1569 (1985)). The formation of the productswas verified by proton NMR by the appearance of a signal at 1.48 ppm forthe methyl protons in analog 11b and a signal at 3.39 ppm for thebenzylic protons in analog 12b. Also observed in the proton NMR was theloss of a signal at 5.84 ppm for the central methylene proton in analog3b. Pedersen (Pedersen et al., Liebigs Ann. Chem. 8, 1557-1569 (1985))observed the monosubstituted product, analog 9b. However, we observedonly the disubstituted product, analog 12b. To verify the formation ofanalogs 11b and 12b, integration of the proton NMR was examined. Thesignal for the benzylic protons at 3.39 ppm was integrated and comparedto each of the alkene signals in the aromatic region. The benzylicsinglet at 3.39 ppm in analog 12b integrates for four protons and thetwo alkene signals in the aromatic region integrate for four protonswhich is to be expected if the product is disubstituted. The sameobservation was made in analog 11b. The methyl singlet at 1.48 ppmintegrates for six protons compared to four protons for the alkenesignals indicating the presence of the disubstituted product. Thestructures were also verified by carbon NMR by the shift of themethylene signal from ˜101.6 ppm to ˜66 ppm and the appearance ofsignals at 21.1 ppm (11b) for the methyl carbons and 37.0 ppm (12b) forthe benzylic carbons. Analog 11b, which is not in the literature, wasverified by elemental analysis.

Eight additional curcumin analogs, 13a, 13b, 14a, 14b, 15a, 15b, 16b,and 17b, were prepared as shown in Scheme 11. These analogs contain twoidentical aryl rings separated by a saturated 7-carbon spacer containingtwo carbonyls. The analogs were designed to test the importance ofsaturation in the spacer. Analogs 13a, 13b, 14a, 14b, 15a, 15b, 16b, and17b were prepared from analogs 3a, 3b, 6a, 6b, 9a, 9b, 11b, and 12brespectively by reduction with palladium on activated carbon under ahydrogen atmosphere on a Parr apparatus following the proceduredescribed by Venkateswarlu (Venkateswarlu et al., Asian J. Chem. 12(1),141-144 (2000)). The formation of the products was verified by protonNMR by the appearance of two multiplets at ˜2.75 ppm for the alkaneprotons in the spacer. Also observed in the proton NMR was the loss oftwo doublets in the aromatic region for the alkene protons. Thestructures were also verified by carbon NMR by the appearance of signalsat ˜30.5 ppm and ˜42.5 ppm for the alkane carbons. The carbon NMR alsoshowed the loss of two signals in the aromatic region for the alkenecarbons. Analogs 14a, 14b, 15a, 15b, and 17b, which are not in theliterature, were verified by high resolution mass spectroscopy. Analog16b, which is not in the literature, was verified by elemental analysis.

Synthesis of 5-Carbon Spacer Analogs

Analogs in series 2, which contain a shorter 5-carbon spacer than incurcumin, were prepared as shown in Schemes 12-27. Analogs 20a-20g, 20i,20k-20ac and 20ae-20ah, as shown in Schemes 12-16, all contain twoidentical aryl rings separated by an unsaturated 5-carbon spacer havinga single carbonyl. These analogs were designed to test the importance ofthe length of the spacer and the type of functional group and locationof the substituent on the aryl ring. Analog 20a, which contains the samearyl substituents as curcumin was prepared as shown in Scheme 12following the procedure as described by Masuda (Masuda et al.,Phytochemistry 32(6), 1557-1560 (1993)).4-Methoxymethyloxy-3-methoxybenzaldehyde (1j) was prepared by reactionof 4-hydroxy-3-methoxybenzaldehyde (1a) with potassium carbonate andchloromethyl methyl ether (18) in a substitution reaction to protect thephenol. Protection was necessary because the aldol reaction on thephenol did not proceed, even upon heating to reflux. The formation ofcompound 1j was verified by proton NMR by the appearance of signals at5.21 ppm for the methylene protons and 3.40 ppm for the methyl protonsof the protecting group.1,5-Bis(4-methoxymethyloxy-3-methoxyphenyl)-1,4-pentadien-3-one (20j)was prepared by reaction of compound 1j with acetone (19) and sodiumhydroxide in an aldol reaction. The formation of the product wasverified by proton NMR by the appearance of a pair of doublets at 7.69ppm and 6.97 ppm with J values of 15.9 Hz for the alkene protons in thespacer. The final step in the preparation of analog 20a was the removalof the groups protecting the phenols. The removal of the protectinggroups was accomplished by reaction of compound 20j with a catalyticamount of concentrated hydrochloric acid in methanol at 65° C. to givethe phenol, analog 20a. The formation of 20a was verified by proton NMRby the loss of signals at 3.40 ppm and 5.21 ppm for the protons of theprotecting group on compound 20j. The structure was also verified bycarbon NMR by the loss of signals at 56.4 ppm and 95.2 ppm for thecarbons of the protecting groups on compound 20j.

Scheme 13 describes the synthesis of analogs 20b-20g, 20i, and 20k-20ac.Analogs 20b-20g, 20i, and 20k-20ac were prepared following the proceduredescribed by Masuda (Masuda et al., Phytochemistry 32(6), 1557-1560(1993)). A substituted benzaldehyde (1b-1g, 1i, and 1k-1ac) was reactedwith acetone (19) and sodium hydroxide in an aldol reaction to giveanalogs 20b-20g, 20i, and 20k-20ac. The formation of the products wasverified by proton NMR by the appearance of a pair of doublets in thearomatic region with J values between 15.6-16.1 Hz for the alkeneprotons present in the spacer. Also observed in the proton NMR was theabsence of a signal at ˜10 ppm for the aldehyde proton of the startingbenzaldehyde (1b-1g, 1i, and 1k-1ac) and a signal at 2.04 ppm for themethyl protons of acetone (19). The structures were also verified bycarbon NMR by the appearance of two signals in the aromatic region forthe alkene carbons in the spacer. Absent from the carbon NMR was asignal at 30.6 ppm for methyl carbons in acetone (19). Analogs 20s and20v, which are not in the literature, were verified by elementalanalysis.

Scheme 14 describes the synthesis of analog 20ae. Analog 20ae wasprepared following the procedure described by White and Zoeller (Whiteet al., U.S. Pat. No. 5,395,692 (1995); Chem. Abstr., 123, P84361n(1995)). 4-Formylbenzoic acid (1ad) was reacted with methanol andthionyl chloride in an esterification reaction to give compound lae. Theformation of the product was verified by proton NMR by the appearance ofa signal at 3.87 ppm for the methyl ester protons. Compound lae was thenreacted with acetone (19) and sodium hydroxide in an aldol reaction togive analog 20ae. The formation of the product was verified by protonNMR by the appearance of a pair of doublets in the aromatic region withJ values of 15.9 Hz and 16.1 Hz for the alkene protons present in thespacer. Also observed in the proton NMR was the loss of a signal at10.12 ppm for the aldehyde proton of the starting benzaldehyde (1ae) anda signal at 2.04 ppm for the methyl protons of acetone (19). Thestructure was also verified by carbon NMR by the appearance of twosignals in the aromatic region for the alkene carbons. The carbon NMRalso showed the loss of a signal at 30.6 ppm for the loss of the methylcarbons of acetone (19).

Scheme 15 describes the synthesis of analog 20af. Analog 20af wasprepared following the procedure described by Royer (Royer et al., J.Med. Chem. 38(13), 2427-2432 (1995)). Analog 20i was demethylated withboron tribromide to give analog 20af. The same reaction was alsoattempted on analogs 20d and 20l-20o with the anticipation of formingthe corresponding tetrahydroxy analogs, however pure stable productscould not be obtained. Immediately following chromatography there was asingle spot on tlc, indicating pure product, however after approximately24 hours, tlc showed a large spot at the origin. In order to verifythese results, the tetramethoxymethyl ether analogs of 20d and 20l weredeprotected using methods described in Scheme 12 (Masuda et al.,Phytochemistry 32(6), 1557-1560 (1993)) to give the correspondingtetrahydroxy analogs. The same results were obtained, thus confirmingthe analogs were decomposing. Analog 20af appeared to be stable and wastested immediately. The formation of analog 20af was verified by protonNMR by the appearance of signals at 9.63 ppm and 9.15 ppm for thephenolic protons. Also observed in the proton NMR was the loss ofsignals at 3.94 ppm and 3.92 ppm for the methyl protons on analog 20i.The structure was also verified by carbon NMR by the loss of a signal at55.9 ppm for the methyl carbons on analog 20i.

Scheme 16 describes the synthesis of analogs 20ag and 20ah. Analogs 20agand 20ah were prepared following the procedure described by Suarez(Suarez et al., World Patent 2004,047,716 (2004); Chem. Abstr., 141,38433 (2004)). Analog 20a or 20f was reacted with acetic anhydride inthe presence of pyridine in an esterification reaction to give analogs20ag and 20ah respectively. The formation of the products was verifiedby proton NMR by the appearence of a signal at 2.31 ppm for the methylprotons of the acetyl groups. The structures were also verified bycarbon NMR by the appearance of a signal at ˜20.9 ppm for the methylcarbons of the acetyl groups and a signal at ˜168.7 ppm for thecarbonyls of the acetyl groups. Analog 20ag and 20ah, which are not inthe literature, were verified by high resolution mass spectroscopy.

Two additional 5-carbon spacer analogs, 23 and 25, were prepared asshown in Scheme 17. Analogs 23 and 25 contain two naphthalene ringsseparated by an unsaturated 5-carbon spacer having a single carbonyl.These analogs were designed to test the importance of naphthalene rings.Compound 22 or 24 was reacted with acetone (19) and sodium hydroxide inan aldol reaction following the procedure described by Masuda (Masuda etal., Phytochemistry 32(6), 1557-1560 (1993)) to give analogs 23 and 25.The formation of the products was verified by proton NMR by theappearance of a pair of doublets in the aromatic region with J valuesbetween 15.7-15.9 Hz for the alkene protons present in the spacer. Alsoobserved in the proton NMR was the loss of a signal at ˜10.25 ppm forthe aldehyde proton of the starting naphthaldehydes (22 and 24) and asignal at 2.04 ppm for the methyl protons of

acetone (19). The structures were also verified by carbon NMR by theappearance of two signals in the aromatic region for the alkene carbonson the spacer. The carbon NMR also showed the loss of a signal at 30.6ppm for the methyl carbons in acetone (19).

Four additional 5-carbon spacer analogs, 28, 29, 31 and 32, wereprepared as shown in Scheme 18. Analogs 28, 29, 31 and 32 contain twonitrogen containing aryl rings separated by an unsaturated 5-carbonspacer having a single carbonyl. These analogs were designed to test theimportance of nitrogen containing aryl rings. Analogs 28 and 31 wereprepared following the procedure described by Zelle and Su (Zelle etal., World Patent 9,820,891 (1998); Chem. Abstr., 129, P23452v (1998)).4-Pyridinecarboxaldehyde (26) or 3-pyridinecarboxaldehyde (30) wasreacted with 1,3-acetonedicarboxylic acid (27) in an aldol type reactionfollowed by addition of concentrated hydrochloric acid to give analogs28 and 31 as hydrochloride salts. Analogs 29 and 32, the free bases,were then prepared by shaking analogs 28 and 31 respectively in sodiumhydroxide. The formation of analogs 28, 29, 31 and 32 were verified byproton NMR by the appearance of a pair of doublets in the aromaticregion with J values between 15.9-16.3 Hz for the alkene protons presentin the spacer. Also observed in the proton NMR was the loss of a signalat ˜10.11 ppm for the aldehyde proton in the startingpyridinecarboxaldehydes (26 and 30) and a signal at 3.55 ppm for themethylene protons of 1,3-acetonedicarboxylic acid (27). The structureswere also verified by carbon NMR by the appearance of two signals in thearomatic region for the alkene carbons and the loss of a signal at˜191.3 ppm for the aldehyde carbon of the startingpyridinecarboxaldehyde (26 and 30). Also observed in the carbon NMR wasthe loss of a signal at 170.3 ppm for the two carboxylic acid carbonsand a signal at 50.1 ppm for the methylene carbons in1,3-acetonedicarboxylic acid (27). The NMR spectra for the unchargedanalogs, 29 and 32 were taken in CDCl₃, whereas the charged analogs 28and 31 were taken in D₂O.

An additional 5-carbon spacer analog, 34, was prepared as shown inScheme 19. Analog 34 contains two sulfur containing aryl rings separatedby an unsaturated 5-carbon spacer having a single carbonyl. This analogwas designed to test the importance of thiophene rings.2-Thiophenecarboxaldehyde (33) was reacted with acetone (19) and sodiumhydroxide in an aldol reaction to give analog 34 following the proceduredescribed by Masuda (Masuda et al., Phytochemistry 32(6), 1557-1560(1993)). The formation of analog 34 was verified by proton NMR by theappearance of a pair of doublets in the aromatic region with J values of15.5 Hz for the alkene protons present in the spacer. Also observed inthe proton NMR was the loss of a signal at 9.79 ppm for the aldehydeproton in the starting 2-thiophenecarboxaldehyde (33) and a signal at2.04 ppm for the methyl protons in acetone (19). The structure was alsoverified by carbon NMR by the appearance of two signals in the aromaticregion for the alkene carbons in the spacer. The carbon NMR also showed

the loss of a signal at 30.6 ppm for the loss of the methyl carbons inacetone (19).

Three additional analogs, 35a, 35e and 35q, were prepared as shown inSchemes 20 and 21. These analogs contain a single aryl ring with anunsaturated 4-carbon tether and a single carbonyl and were designed totest the necessity of two aryl rings. Analog 35a was prepared as shownin Scheme 20 following the procedure described by Masuda (Masuda et al.,Phytochemistry 32(6), 1557-1560 (1993)). Compound 1j, prepared aspreviously reported in Scheme 12, was reacted with excess acetone (19)and sodium hydroxide in an aldol reaction to give compound 35j.Protection was necessary because the aldol reaction on the phenol didnot proceed, even upon heating to reflux. The formation of compound 35jwas verified by proton NMR by the appearance of a pair of doublets inthe aromatic region with J values of 16.1 Hz for the alkene protonspresent on the tether and a signal at 2.34 ppm for the methyl protonspresent on the tether. Also observed in the proton NMR was the loss of asignal at 9.75 ppm for the aldehyde proton in the starting benzaldehyde(1j). Compound 35j was then reacted with a catalytic amount ofconcentrated hydrochloric acid to give the phenol, analog 35a. Theformation of analog 35a was verified by proton NMR by the loss ofsignals at 3.48 ppm and 5.24 ppm for the protons of the protecting groupin compound 35j. The structure was also verified by carbon NMR by theloss of signals at

56.2 ppm and 94.8 ppm for the carbons of the protecting group incompound 35j.

Scheme 21 describes the synthesis of analogs 35e and 35q following theprocedure described by Masuda (Masuda et al., Phytochemistry 1993,32(6), 1557-1560). Compound 1e or 1q was reacted with excess acetone(19) and sodium hydroxide in an aldol reaction to give analogs 35e and35q. The formation of the products was verified by proton NMR by theappearance of a pair of doublets in the aromatic region with J values of16.3-16.5 Hz for the alkene protons present on the tether and theappearance of a signal at ˜2.37 ppm for the methyl protons on thetether. Also observed in the proton NMR was the loss of a signal at˜9.94 ppm for the aldehyde proton in the starting benzaldehyde (1e or1q) and the loss of a signal at 2.04 ppm for the methyl protons ofacetone (19). The structures were also verified by carbon NMR by theappearance of two signals in the aromatic region for the alkene carbonspresent on the tether.

Two additional 5-carbon spacer analogs, 36a and 36e, were prepared asshown in Schemes 22 and 23. These analogs contain two different arylrings separated by a 5-carbon unsaturated spacer containing a singlecarbonyl and were designed to test the importance of symmetry in analogswith a 5-carbon spacer. Analog 36a was prepared as shown in Scheme 22following the procedure described by Masuda (Masuda et al.,Phytochemistry 32(6), 1557-1560 (1993)). Compound 35j, prepared as shownin Scheme 21, was reacted with benzaldehyde (1b) in an aldol reaction togive compound 36j. The formation of compound 36j was verified by protonNMR by the appearance of a second pair of doublets in the aromaticregion for the new alkene in the spacer and the loss of a signal at 2.34ppm for the methyl protons on the tether in compound 35j. Compound 36jwas then reacted with a catalytic amount of concentrated hydrochloricacid to give the phenol, analog 36a. The formation of analog 36a wasverified by proton NMR by the loss of signals at 3.5 ppm and 5.2 ppm forthe protons of the protecting group in compound 36j. The structure wasalso verified by carbon NMR by the loss of signals at 56.2 ppm and 94.8ppm for the carbons of the protecting group in compound 36j.

Scheme 23 describes the synthesis of analog 36e. Analog 35e, prepared asshown in Scheme 21, was reacted with benzaldehyde (1b) and sodiumhydroxide in an aldol reaction following the procedure described byMasuda (Masuda et al., Phytochemistry 32(6), 1557-1560 (1993)) to giveanalog 36e. The formation of the product was verified by proton NMR bythe appearance of a second pair of doublets in the aromatic region withJ values of 15.9-16.1 Hz for the new alkene protons present in thespacer and the loss of a signal at 2.34 ppm for the methyl protons onthe tether in analog 35e. The structure was also verified by carbon NMRby the appearance of two signals in the aromatic region for the newalkene carbons and the loss of a signal at 27.5 ppm for the methylcarbon on the tether in analog 35e.

Two additional 5-carbon spacer analogs, 38a and 38b, were prepared asshown in Schemes 24 and 25. These analogs contain two identical arylrings separated by an unsaturated 5-carbon spacer having a singlecarbonyl and a saturated ring. Analogs 38a and 38b were designed to testthe importance of a ring in the spacer. Analog 38a was prepared as shownin Scheme 24 following the procedure described by Masuda (Masuda et al.,Phytochemistry 32(6), 1557-1560 (1993)). Compound 1j, prepared as shownin Scheme 12, was reacted with cyclohexanone (37) and sodium hydroxidein an aldol reaction to give compound 38j. The formation of compound 38jwas verified by proton NMR by the appearance of a signal at 7.74 ppm forthe alkene protons on the spacer and the loss of a signal at 9.75 ppmfor the aldehyde proton in the starting benzaldehyde (1j). Compound 38jwas then reacted with a catalytic amount of concentrated hydrochloricacid to give the phenol, analog 38a. The formation of the product wasverified by proton NMR by appearance of a signal at 5.88 ppm for thephenolic protons and the loss of signals at 3.52 ppm and 5.26 ppm forthe

protons of the protecting group in compound 38j. The structure was alsoverified by carbon NMR by the loss of signals at 55.8 ppm and 95.1 ppmfor the carbons of the protecting group in compound 38j.

Scheme 25 describes the synthesis of analog 38b following the proceduredescribed by Masuda (Masuda et al., Phytochemistry 32(6), 1557-1560(1993)). Benzaldehyde (1b) was reacted with cyclohexanone (37) andsodium hydroxide in an aldol reaction to give analog 38b. The formationof the product was verified by proton NMR by the appearance of a signalat 7.80 ppm for the alkene protons on the spacer and the loss of asignal at 9.94 ppm for the aldehyde proton on the starting benzaldehyde(1b). The structure of the product was also verified by carbon NMR bythe appearance of two signals in the aromatic region for the alkenecarbons on the spacer.

Two additional 5-carbon spacer analogs, 39b and 40b, were prepared asshown in Scheme 26 following the procedure described by Venkateswarlu(Venkateswarlu et al., Asian J. Chem. 12(1), 141-144 (2000)). Analog 39bcontains two identical aryl rings separated by a saturated 5-carbonspacer and was designed to test the importance of unsaturation in thespacer of series 2 analogs. Analog 40b was designed to test theimportance of a carbonyl in the spacer. Analogs 39b and 40b wereprepared by reduction of analog 20b with palladium on activated carbonunder a hydrogen atmosphere on a Parr apparatus. A mixture containinganalogs 39b and 40b was obtained and separated by chromatography. Theformation of analog 39b was verified by proton NMR by the appearance oftriplets at 2.76 ppm and 2.97 ppm for the methylene protons on thespacer. The proton NMR also showed the loss of a pair of doublets in thearomatic region for the alkene protons. The structure was also verifiedby carbon NMR by the appearance of signals at 29.6 ppm and 44.2 ppm forthe methylene carbons on the spacer and the loss of two signals in thearomatic region for the alkene carbons on the spacer in analog 20b. Theformation of analog 40b was verified by proton NMR by the appearance ofa pentet for the proton on the carbon bearing the hydroxyl group andmultiplets at 1.85 ppm and 2.77 ppm for the methylene protons on thespacer. The structure was also verified by carbon NMR by the appearanceof signals at 32.1 ppm, 39.2 ppm and 70.8 ppm for the carbon bearing thehydroxyl group and for the methylene carbons on the spacer. The carbonNMR also shows the loss of two signals in the aromatic region for thealkene carbons on the spacer and the loss of a signal at 188.7 ppm forthe carbonyl carbon in analog 20b.

Two additional 5-carbon spacer analogs, 42b and 43b, were prepared asshown in Scheme 27 following the procedure described by Yadav and Kapoor(Yadav et al., Tetrahedron 52(10), 3659-3668 (1996)). These analogscontain two identical aryl rings separated by a saturated 5-carbonspacer containing both a carbonyl and two epoxide rings. These analogswere designed to test the importance of epoxide rings on the spacer.Analogs 42b and 43b were prepared by reaction of analog 20b with t-butylhydroperoxide and aluminum oxide-potassium fluoride in an epoxidationreaction. A mixture containing analog 42b and analog 43b was formed andthe trans/trans isomer, analog 42b, was separated from the cis/cisisomer, analog 43b, through recrystallization from ethanol as describedby Yadav and Kapoor (Yadav et al., Tetrahedron 52(10), 3659-3668(1996)). The formation of the products was verified by proton NMR by theappearance of a pair of doublets at 3.30 ppm and 4.09 ppm for the alkaneprotons on the spacer in analog 42b. Analog 43b has a pair of doubletsat 3.72 ppm and 4.18 ppm for the alkane protons on the spacer. Alsoobserved in the proton NMR was the loss of two signals in the aromaticregion for the alkene protons on the spacer in analog 20b. Thestructures were also verified by carbon NMR by the appearance of a twosignals at ˜59.9 ppm for the alkane carbons on the spacer. The carbonNMR also showed the loss of two signals in the aromatic region for thealkene carbons on the spacer in analog 20b.

Synthesis of 3-Carbon Spacer Analogs

Analogs in series 3, which contain a 3-carbon spacer, were prepared asshown in Schemes 28-37. Analogs 45a and 45b contain two identical arylrings separated by an unsaturated 3-carbon spacer having a singlecarbonyl and were designed to test the importance of the length of thespacer. Analog 45a was prepared as shown in Scheme 28 following theprocedures described by Masuda (Masuda et al., Phytochemistry 32(6),1557-1560 (1993)) and by Kohler and Chadwell (Kohler et al., Org.Synth., Coll. Vol. 178-80 (1932)). 4-Hydroxy-3-methoxyacetophenone (44a)was reacted with potassium carbonate and chloromethyl methyl ether (18)in a substitution reaction to give compound 44j. The formation of theproduct was verified by proton NMR by the appearance of signals at 3.33ppm and 5.12 ppm for the protons of the protecting group. Compound 1j,prepared as shown in Scheme 12, was reacted with compound 44j and bariumhydroxide in an aldol reaction to give compound 45j. The formation ofthe product was verified by proton NMR by the appearance of a pair ofdoublets in the aromatic region with J values of 15.5 Hz for the alkeneprotons on the spacer. Also observed in the proton NMR was the loss of asignal at 2.38 ppm for the methyl protons of the starting acetophenone(44j) and the loss of a signal at 9.75 ppm for the aldehyde proton ofthe starting benzaldehyde (1j). Compound 45j was then reacted with acatalytic amount of concentrated hydrochloric acid to give the phenol,analog 45a. The formation of the product was verified by proton NMR bythe appearance of signals at 6.00 ppm and 6.19 ppm for the phenolicprotons. Also observed in the proton NMR was the loss of signals at 3.50ppm, 5.25 ppm

and 5.30 ppm for the protons in the protecting groups in compound 45j.The structure was also verified by carbon NMR by the loss of signals at˜57 ppm and ˜95 ppm for the carbons of the protecting groups in compound45j.

Scheme 29 describes the synthesis of analog 45b following the proceduredescribed by Kohler and Chadwell (Kohler et al., Org. Synth., Coll. Vol.178-80 (1932)). Acetophenone (44b) was reacted with benzaldehyde (1b)and sodium hydroxide in an aldol reaction to give analog 45b. Theformation of the product was verified by proton NMR by the appearance ofa pair of doublets in the aromatic region with J values of 15.7 Hz forthe alkene protons on the spacer. Also observed in the proton NMR wasthe loss of a signal at 9.74 ppm for the aldehyde proton on the startingbenzaldehyde (1b) and a signal at 2.51 ppm for the methyl protons in thestarting acetophenone (44b). The structure was also verified by carbonNMR by the appearance of two signals in the aromatic region for thealkene carbons on the spacer and the loss of a signal at 26.0 ppm forthe methyl carbon on the starting acetophenone (44b).

Six additional 3-carbon spacer analogs, 46a, 46ak-46am, 48a and 48ad,were prepared as shown in Schemes 30-34. Analogs 46a, 46ak-46am, 48a and48ad contain two different aryl rings separated by an unsaturated3-carbon spacer having a single carbonyl. These analogs were designed totest the importance of the length of the spacer and the importance ofring symmetry in series 3 analogs. Analog 46a was prepared as shown inScheme 30 following the procedures described by Masuda (Masuda et al.,Phytochemistry 32(6), 1557-1560 (1993)) and by Kohler and Chadwell(Kohler et al., Org. Synth., Coll. Vol. 178-80 (1932)). Compound 44j,prepared as shown in Scheme 28, was reacted with benzaldehyde (1b) andbarium hydroxide in an aldol reaction to give compound 46j. Theformation of the product was verified by proton NMR by the appearance ofa pair of doublets in the aromatic region with J values of 15.7 Hz forthe alkene protons on the spacer. Also observed in the proton NMR wasthe loss of a signal at 2.38 ppm for the methyl protons on the startingacetophenone (44j) and a signal at 9.74 ppm for the aldehyde proton inthe starting benzaldehyde (1b). Compound 46j was then reacted with acatalytic amount of concentrated hydrochloric acid to give the phenol,analog 46a. The formation of the product was verified by proton NMR bythe appearance of a signal at 6.29 ppm for the phenolic proton. Alsoobserved in the proton NMR was the loss of signals at 3.48 ppm and 5.28ppm for the protons of the protecting group in compound 46j. Thestructure was also verified by carbon NMR by the loss of signals at ˜57ppm and ˜95 ppm for the carbons of the protecting group in compound 46j.

Scheme 31 describes the synthesis of analogs 46ak and 46al following theprocedure described by Kohler and Chadwell (Kohler et al., Org. Synth.,Coll. Vol. 178-80 (1932)). Acetophenone 44ak or 44al was reacted withbenzaldehyde (1b) and barium hydroxide in an aldol reaction to giveanalogs 46ak or 46al respectively. The formation of the products wasverified by proton NMR by the appearance of a pair of doublets in thearomatic region with J values of 15.9-16.1 Hz for the alkene protons onthe spacer. Also observed in the proton NMR was the loss of a signal at9.74 ppm for the aldehyde proton in the starting benzaldehyde (1b) and asignal at ˜2.49 ppm for the methyl protons of the starting acetophenones(44ak and 44al). The structure of the product was also verified bycarbon NMR by the appearance of two signals in the aromatic region forthe alkene carbons on the spacer. Also observed in the carbon NMR wasthe loss of a signal at 26.7 ppm for the methyl carbon on the startingacetophenones (44ak and 44al).

Scheme 32 describes the synthesis of analog 46ad following the proceduredescribed by Cleeland (Cleeland et al., U.S. Pat. No. 4,045,487 (1977);Chem. Abstr., 87, P167872u (1977)). Compound 44al was reacted withconcentrated sulfuric acid in a hydrolysis reaction to give compound44ad. The formation of the product was verified by proton NMR by theappearance of a signal at 13.34 ppm for the carboxylic acid proton.Compound 44ad was then reacted with benzaldehyde (1b) and sodiumhydroxide in an aldol reaction followed by acidification with dilutehydrochloric acid to give analog 46ad. The formation of the product wasverified by proton NMR by the appearance of a pair of doublets in thearomatic region with J values of 15.5-16.1 Hz for the alkene protons onthe spacer. Also observed in the proton NMR was the loss of a signal at2.43 ppm for the methyl protons on the starting acetophenone (44ad) anda signal at 9.74 ppm for the aldehyde proton in the startingbenzaldehyde (1b). The structure of the product was also verified bycarbon NMR by the appearance of two signals in the aromatic region forthe alkene carbons on the spacer. Also seen in the carbon NMR was theloss of a signal at 23.7 ppm for the methyl carbon in the startingacetophenone (44ad).

Scheme 33 describes the synthesis of analog 48a following the proceduredescribed by Takagaki (Takagaki et al., European Patent 370,461 (1990);Chem. Abstr., 113, P230963x (1990)). Compound 1a was reacted with3,4-dihydropyran and pyridiniump-toluenesulfonate in a substitutionreaction to give compound lam. The formation of the product was verifiedby proton NMR by the appearance of multiplets in the aliphatic regionfor the protecting group protons. Compound lam was then reacted withacetophenone (44b) and barium hydroxide in an aldol reaction to givecompound 48am. The formation of the product was verified by proton NMRby the appearance of a pair of doublets in the aromatic region with Jvalues of 15.5-15.9 Hz for the alkene protons on the spacer. Alsoobserved in the proton NMR was the loss of a signal at 2.38 ppm for themethyl protons on the starting acetophenone (44b) and a signal at 9.87ppm for the aldehyde proton in the starting benzaldehyde (1am). Compound48am was then reacted with p-toluenesulfonic acid to give the phenol,analog 48a. The formation of the product was verified by proton NMR bythe appearance of a signal at 5.96 ppm for the phenolic proton. Alsoobserved in the proton NMR was the loss of multiplets in the aliphaticregion for the protons of the protecting group in compound 48am. Thestructure was also verified by carbon NMR by the loss of five signals inthe aliphatic region for the carbons of the protecting group in compound48am.

Scheme 34 describes the synthesis of analog 48ad following the proceduredescribed by Cleeland (Cleeland et al., U.S. Pat. No. 4,045,487 (1977);Chem. Abstr., 87, P167872u (1977)). Compound lad was reacted withcompound 44b and sodium hydroxide in an aldol reaction followed byacidification with dilute hydrochloric acid to give analog 48ad. Theformation of the product was verified by proton NMR by the appearance ofa pair of doublets in the aromatic region for the alkene protons on thespacer. Also observed in the proton NMR was the loss of a signal at 2.38ppm for the methyl protons on the starting acetophenone (44b) and asignal at 10.12 ppm for the

aldehyde proton in the starting benzaldehyde (1ad). The structure of theproduct was also verified by carbon NMR by the appearance of two signalsin the aromatic region for the alkene carbons on the spacer. Also seenin the carbon NMR was the loss of a signal at 26.0 ppm for the methylcarbon in the starting acetophenone (44b).

An additional 3-carbon spacer analog, 50b, was prepared as shown inScheme 35 following the procedure described by Chisolm (Chisolm et al.,Patent 050,713 (1992); Chem. Abstr., 115, P207660d (1992)). Analog 50bcontains two aryl rings separated by a 3-carbon spacer having twocarbonyls. This analog was designed to test the importance of twocarbonyls in a 3-carbon spacer. Acetophenone (44b) was reacted withmethyl benzoate (49) and sodium methoxide in a condensation reaction togive analog 50b. The formation of the product was verified by proton NMRby the appearance of a signal at 6.85 ppm for the enol proton on thespacer and the loss of a signal at 2.38 for the methyl protons onacetophenone (44b) and a signal at 3.88 ppm for the methyl ester protonsof methyl benzoate (49). The structure was also verified by carbon NMRby the appearance of signals at 93.1 ppm for the enol carbon and 185.6ppm for the carbonyl carbons on the spacer. Also observed in the carbonNMR was the loss of signals at 166.2 ppm for the carbonyl carbon and51.4 ppm for the methyl carbon on methyl benzoate (49) and signals at197.3 ppm for the carbonyl carbon and 26.0 for the methyl carbon onacetophenone (44b).

Six additional analogs, 52b, 52c, 52e, 52aa, 52ac and 53 were preparedas shown in Schemes 36 and 37 following the procedure described bySelvaraj (Selvaraj et al., Ind. J. Chem., Sect. B 26B, 1104-1105(1987)). Analogs 52b, 52c, 52e, 52aa, 52ac and 53 contain two identicalaryl rings separated by a 3-carbon spacer having both a carbonyl and asaturated heterocyclic ring and were designed to test the importance ofa heterocyclic ring in the spacer. Analogs 52b, 52c, 52e, 52aa and 52acwere prepared as shown is Scheme 36 by reaction of analogs 20b, 20c,20e, 20aa and 20ac with methylamine (51) in a Michael addition reaction.The formation of the products was verified by proton NMR by theappearance of a pair of doublets at ˜2.50 ppm and ˜3.45 ppm for theprotons alpha to the carbonyl and a triplet at ˜2.82 ppm for the protonsalpha to the amine. The structures were also verified by carbon NMR bythe appearance of signals at ˜50.8 ppm and ˜70.2 ppm for the alkanecarbons in the nitrogen containing heterocyclic ring and by the loss oftwo signals in the aromatic region for the alkene carbons. Analogs 52cand 52ac, which are not in the literature, were verified by highresolution mass spectroscopy.

Scheme 37 describes the synthesis of analog 53 following the proceduredescribed by Selvaraj (Selvaraj et al., Ind. J. Chem., Sect. B 26B,1104-1105 (1987)). Analog 25 was reacted with methylamine (51) in aMichael addition reaction to give analog 53. The formation of theproducts was verified by proton NMR by the appearance of a pair ofdoublets at 2.59 ppm and 3.66 ppm for the protons alpha to the carbonyland a triplet at 2.97 ppm for the protons alpha to the amine. Thestructures were also verified by carbon NMR by the appearance of signalsat 50.7 ppm and 70.3 ppm for the alkane carbons in the nitrogencontaining heterocyclic ring and by the loss of two signals in thearomatic region for the alkene carbons. Analog 53, which is not in theliterature, was verified by high resolution mass spectroscopy.

Experimental

Reagent quality solvents were used without purification with theexception of ethyl acetate which was distilled from magnesium sulfatebefore use. Liquid benzaldehydes, acetone and acetyl acetone weredistilled before use. All other reagents were obtained from commercialsources and used without further purification. All compounds isolatedwere greater than 95% pure by proton and carbon NMR. Columnchromatographic separations were performed using EM Science type 60silica gel (230-400 mesh). Melting points were determined on a ThomasHoover capillary melting point apparatus and are uncorrected. NMRspectra were recorded on a Bruker AC250 (250 MHz) NMR spectrometer inCDCl₃ unless otherwise noted. Chemical shifts are reported in ppm (δ)relative to CDCl₃ at 7.24 ppm for proton NMR and 77.0 for carbon NMR orDMSO at 2.49 ppm for proton NMR and 39.5 ppm for carbon NMR. Proton NMRpeaks are reported as follows: chemical shift, multiplicity (s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, dd=doublet of doublets anddt=doublet of triplets), integration, and coupling constants (J in Hz).High resolution mass spectra were performed at the UNM Mass SpectrometryFacility, University of New Mexico, Albuquerque N.Mex. Analytical datawas obtained from Galbraith Laboratories, Knoxville Tenn.

4-Methoxymethyloxy-3-methoxybenzaldehyde (1j).4-Hydroxy-3-methoxybenzaldehyde (1a, 2.00 g, 13.1 mmol) and potassiumcarbonate (9.00 g, 65.1 mmol) were combined in dimethyl formamide (30ml) and stirred for 15 min at room temperature. Chloromethyl methylether (1.60 ml, 21.1 mmol) was added and stirring was continued for 6 hrat room temperature. The resulting mixture was filtered and the filtrateextracted into ethyl acetate, washed with saturated sodium chloride,dried over magnesium sulfate, filtered and evaporated to give 2.55 g(99%) of a white solid: mp 39-40° C. [expected mp 41° C.]; ¹H NMR: δ3.40 (s, 3H), 3.83 (s, 3H), 5.21 (s, 2H), 7.15 (d, 1H, J=8.7 Hz), 7.30(dd, 1H, J=6.0, 2.0 Hz), 7.32 (s, 1H), 9.75 (s, 1H); ¹³C NMR: δ 55.8,56.2, 94.8, 109.4, 114.6, 125.9, 130.9, 149.8, 151.7, 190.4.

4-Carbmethoxybenzaldehyde (1ae). 4-Formylbenzoic acid (1ad, 1.00 g, 6.7mmol) was dissolved in dry methanol (200 ml) and stirred for 10 min at0° C. Thionyl chloride (6 ml, 82.3 mmol) was added dropwise and themixture stirred for 90 min at 0° C. and 3 hr at room temperature. Themethanol was evaporated and the resulting residue extracted intodichloromethane, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford a solid. The crudesolid was recrystallized from hexane to give 1.07 g (98%) of a whitesolid: mp 60-62° C. [expected mp 61° C.]; ¹H NMR: δ 3.87 (s, 3H), 8.01(d, 2H, J=7.9 Hz), 8.13 (d, 2H, J=7.6 Hz), 10.08 (s, 1H); ¹³C NMR: δ52.7, 129.7, 129.9, 134.4, 139.1, 165.6, 192.9.

1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (3a). Boricanhydride (0.49 g, 7.0 mmol) was combined with 2,4-pentanedione (2, 1.05ml, 10.0 mmol) and stirred for 18 hr at room temperature under anitrogen atmosphere. A solution of dry ethyl acetate (10 ml),4-hydroxy-3-methoxybenzaldehyde (1a, 3.04 g, 20.0 mmol) and tributylborate (11.00 ml, 40.5 mmol) was added and the mixture stirred for 15min at room temperature. Butylamine (0.20 ml, 2.0 mmol) was addeddropwise over 30 min and stirring was continued for 18 hr at roomtemperature. Hydrochloric acid (15 ml, 0.4 N) was warmed to 60° C.,added to the mixture and stirring was continued for 1 hr. The resultingmixture was extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a solid. The crude solid was triturated with methanol to give2.82 g (77%) of an orange-yellow solid: mp 182-184° C. [expected mp182-183° C.]; ¹H NMR: (DMSO) δ 3.83 (s, 6H), 6.05 (s, 1H), 6.74 (d, 2H,J=15.9 Hz), 6.82 (d, 2H, J=8.1 Hz), 7.14 (d, 2H, J=8.0 Hz), 7.31 (s,2H), 7.54 (d, 2H, J=15.7 Hz), 9.63 (s, 2H), 16.29 (s, 1H); ¹³C NMR:(DMSO) δ 55.6, 100.5, 111.3, 115.5, 120.9, 122.8, 126.2, 140.4, 147.8,149.1, 182.8.

1,7-Diphenyl-1,6-heptadiene-3,5-dione (3b). Boric anhydride (0.49 g, 7.0mmol) was combined with 2,4-pentanedione (2, 1.05 ml, 10.0 mmol) andstirred for 18 hr at room temperature under a nitrogen atmosphere. Asolution of dry ethyl acetate (10 ml), benzaldehyde (1b, 2.05 g, 20.2mmol) and tributyl borate (11.00 ml, 40.5 mmol) was added and themixture stirred for 15 min at room temperature. Butylamine (0.20 ml, 2.0mmol) was added dropwise over 30 min and stirring was continued for 18hr at room temperature. Hydrochloric acid (15 ml, 0.4 N) was warmed to60° C., added to the mixture and stirring was continued for 1 hr. Theresulting mixture was extracted into ethyl acetate, washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford a solid. The crude solid was triturated withmethanol to give 0.90 g (33%) of a yellow solid: mp 140-142° C.[expected mp 139-140° C.]; ¹H NMR: δ 5.84 (s, 1H), 6.62 (d, 2H, J=15.7Hz), 7.39 (m, 6H), 7.54 (dd, 4H, J=7.4, 4.0 Hz), 7.66 (d, 2H, J=15.9Hz), 15.85 (s, 1H); ¹³C NMR: δ 101.6, 124.1, 128.0, 128.9, 130.0, 135.0,140.5, 183.2.

1,7-Bis(2-methoxyphenyl)-1,6-heptadiene-3,5-dione (3c). Boric anhydride(0.33 g, 4.7 mmol) was combined with 2,4-pentanedione (2, 0.70 ml, 6.7mmol) and stirred for 18 hr at room temperature. A solution of dry ethylacetate (15 ml), 2-methoxybenzaldehyde (1c, 1.81 g, 13.3 mmol) andtributyl borate (7.25 ml, 26.7 mmol) was added and the mixture stirredfor 15 min at room temperature. Butylamine (1.00 ml, 10.1 mmol) wasadded dropwise over 30 min and stirring was continued for 18 hr at roomtemperature. Hydrochloric acid (15 ml, 0.4 N) was warmed to 60° C.,added to the mixture and stirring was continued for 1 hr. The resultingmixture was extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a semi-solid. The crude semi-solid was chromatographed on silicagel with ethyl acetate/hexane to give 0.48 g (21%) of a yellow crystals:mp 121-123° C. [expected mp 121-122° C.]; ¹H NMR: δ 3.89 (s, 6H), 5.86(s, 1H), 6.71 (d, 2H, J=16.1 Hz), 6.94 (m, 4H), 7.33 (dt, 2H, J=8.1, 1.4Hz), 7.54 (dd, 2H, J=7.8, 1.4 Hz), 7.97 (d, 2H, J=16.1 Hz), 16.00 (s,1H); ¹³C NMR: δ 55.5, 101.4, 111.1, 120.6, 124.0, 124.7, 128.5, 131.1,135.6, 158.3, 183.6.

1,7-Bis(2,3-dimethoxyphenyl)-1,6-heptadiene-3,5-dione (3d). Boricanhydride (0.49 g, 7.0 mmol) was combined with 2,4-pentanedione (2, 1.05ml, 10.0 mmol) and stirred for 18 hr at room temperature under anitrogen atmosphere. A solution of dry ethyl acetate (10 ml),2,3-dimethoxybenzaldehyde (1d, 3.32 g, 20.0 mmol) and tributyl borate(11.00 ml, 40.5 mmol) was added and the mixture stirred for 15 min atroom temperature. Butylamine (0.20 ml, 2.0 mmol) was added dropwise over30 min and stirring was continued for 18 hr at room temperature.Hydrochloric acid (15 ml, 0.4 N) was warmed to 60° C., added to themixture and stirring was continued for 1 hr. The resulting mixture wasextracted into ethyl acetate, washed with saturated sodium chloride,dried over magnesium sulfate, filtered and evaporated to afford asemi-solid. The crude semi-solid was triturated with methanol to give2.01 g (51%) of a yellow solid: mp 117-120° C. [expected mp 117-120°C.]; ¹H NMR: δ 3.87 (s, 12H), 5.87 (s, 1H), 6.68 (d, 2H, J=16.1 Hz),6.92 (d, 2H, J=8.2 Hz), 7.05 (t, 2H, J=8.0 Hz), 7.18 (d, 2H, J=6.8 Hz),7.95 (d, 2H, J=16.1 Hz), 15.88 (s, 1H); ¹³C NMR: (DMSO) δ 55.7, 60.7,101.9, 114.6, 118.7, 124.1, 125.1, 128.0, 134.2, 147.7, 152.6, 182.9;Anal. Calcd for C₂₃H₂₄O₆: C, 69.68; H, 6.10. Found: C, 69.43; H, 6.16.

1,7-Bis(4-methoxyphenyl)-1,6-heptadiene-3,5-dione (3e). Boric anhydride(0.49 g, 7.0 mmol) was combined with 2,4-pentanedione (2, 1.05 ml, 10.0mmol) and stirred for 18 hr at room temperature under a nitrogenatmosphere. A solution of dry ethyl acetate (10 ml),4-methoxybenzaldehyde (1e, 2.43 ml, 20.0 mmol) and tributyl borate(11.00 ml, 40.5 mmol) was added and the mixture stirred for 15 min atroom temperature. Butylamine (0.20 ml, 2.0 mmol) was added dropwise over30 min and stirring was continued for 18 hr at room temperature.Hydrochloric acid (15 ml, 0.4 N) was warmed to 60° C., added to themixture and stirring was continued for 1 hr. The resulting mixture wasextracted into ethyl acetate, washed with saturated sodium chloride,dried over magnesium sulfate, filtered and evaporated to afford a solid.The crude solid was triturated with methanol to give 2.83 g (84%) of ayellow solid: mp 157-159° C. [expected mp 154-155° C.]; ¹H NMR: δ 3.82(s, 6H), 5.75 (s, 1H), 6.48 (d, 2H, J=15.9 Hz), 6.90 (d, 4H, J=8.7 Hz),7.49 (d, 4H, J=8.7 Hz), 7.60 (d, 2H, J=15.9 Hz), 16.04 (s, 1H); ¹³C NMR:δ 55.4, 101.2, 114.4, 121.9, 127.9, 129.7, 140.0, 161.2, 183.2.

1,7-Bis(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (3f). Boric anhydride(0.33 g, 4.7 mmol) was combined with 2,4-pentanedione (2, 0.70 ml, 6.7mmol) and stirred for 18 hr at room temperature. A solution of dry ethylacetate (15 ml), 4-hydroxybenzaldehyde (1f, 1.62 g, 13.3 mmol) andtributyl borate (7.25 ml, 26.7 mmol) was added and the mixture stirredfor 15 min at room temperature. Butylamine (1.0 ml, 10.1 mmol) was addeddropwise over 30 min and stirring was continued for 18 hr at roomtemperature. Hydrochloric acid (15 ml, 0.4 N) was warmed to 60° C.,added to the mixture and stirring was continued for 1 hr. The resultingmixture was extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a solid. The crude solid was recrystallized from methanol to give0.40 g (19%) of red-orange crystals: mp 226-228° C. [expected mp223-224° C.]; ¹H NMR: (DMSO) δ 6.03 (s, 1H), 6.67 (d, 2H, J=15.9 Hz),6.81 (d, 4H, J=7.7 Hz), 7.55 (m, 6H), 10.03 (s, 2H), 16.37 (s, 1H); ¹³CNMR: (DMSO) δ 100.7, 115.8, 120.7, 125.7, 130.1, 140.1, 159.5, 182.9.

1,7-Bis(4-dimethylaminophenyl)-1,6-heptadiene-3,5-dione (3g). Boricanhydride (0.33 g, 4.7 mmol) was combined with 2,4-pentanedione (2, 0.70ml, 6.7 mmol) and stirred for 18 hr at room temperature. A solution ofdry ethyl acetate (15 ml), 4-dimethylaminobenzaldehyde (1g, 2.00 g, 13.4mmol) and tributyl borate (7.25 ml, 26.7 mmol) was added and the mixturestirred for 15 min at room temperature. Butylamine (1.0 ml, 10.1 mmol)was added dropwise over 30 min and stirring was continued for 18 hr atroom temperature. Hydrochloric acid (15 ml, 0.4 N) was warmed to 60° C.,added to the mixture and stirring was continued for 1 hr. The resultingmixture was extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a solid. The crude solid was triturated with methanol to give0.57 g (23%) of a purple solid: mp 207-208° C. [expected mp 210-212°C.]; ¹H NMR: (DMSO) δ 3.01 (s, 12H), 5.71 (s, 1H), 6.41 (d, 2H, J=15.61Hz), 6.67 (d, 4H, J=8.02 Hz), 7.44 (d, 4H, J=8.11 Hz), 7.58 (d, 2H,J=15.65 Hz), 16.56 (s, 1H); ¹³C NMR: (DMSO) δ 39.6, 100.3, 111.7, 118.5,122.0, 129.7, 140.3, 151.4, 182.6.

1,7-Bis(3-hydroxy-4-methoxyphenyl)-1,6-heptadiene-3,5-dione (3h). Boricanhydride (0.49 g, 7.0 mmol) was combined with 2,4-pentanedione (2, 1.05ml, 10.0 mmol) and stirred for 18 hr at room temperature under anitrogen atmosphere. A solution of dry ethyl acetate (10 ml),3-hydroxy-4-methoxybenzaldehyde (1h, 3.04 g, 20.0 mmol) and tributylborate (1.0 ml, 40.5 mmol) was added and the mixture stirred for 15 minat room temperature. Butylamine (0.20 ml, 2.0 mmol) was added dropwiseover 30 min and stirring was continued for 18 hr at room temperature.Hydrochloric acid (15 ml, 0.4 N) was warmed to 60° C., added to themixture and stirring was continued for 1 hr. The resulting mixture wasfiltered to afford a solid. The crude solid was triturated with methanolto give 2.60 g (71%) of a orange-yellow solid: mp 190-192° C. [expectedmp 189-190° C.]; ¹H NMR: (DMSO) δ 3.78 (s, 6H), 6.09, (s, 1H), 6.60 (d,2H, J=15.9 Hz), 6.49 (d, 2H, J=8.9 Hz), 7.11 (m, 4H), 7.47 (d, 2H,J=15.9 Hz), 9.19 (s, 2H); ¹³C NMR: (DMSO) δ 55.6, 100.9, 112.0, 114.0,121.2, 121.5, 127.5, 140.2, 146.6, 149.8, 182.8.

1,7-Bis(3,4-dimethoxyphenyl)-1,6-heptadiene-3,5-dione (3i). Boricanhydride (0.49 g, 7.0 mmol) and 2,4-pentanedione (2, 1.05 ml, 10.0mmol) were combined in dry ethyl acetate (10 ml) and stirred for 30 minat 40° C. 3,4-Dimethoxybenzaldehyde (1i, 3.32 g, 20.0 mmol) and tributylborate (7.90 ml, 29.1 mmol) were added and stirring was continued for 30min at 40° C. A solution of butylamine (1.5 ml, 15.2 mmol) in dry ethylacetate (10 ml) was added dropwise over 15 min and stirring wascontinued for 18 hr at 40° C. Hydrochloric acid (10 ml, 2 N) was addedand the mixture stirred for 1 hr at 60° C. The resulting mixture wascooled to room temperature, extracted with ethyl acetate, washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford a semi-solid. The crude semi-solid waschromatographed on silica gel with ethyl acetate/hexane to give a solid.The crude solid was recrystallized from methanol to give 1.16 g (29%) ofan orange solid: mp 129-131° C. [expected mp 128-130° C.]; ¹H NMR:(DMSO) δ 3.79 (s, 6H), 3.81 (s, 6H), 6.10 (s, 1H), 6.82 (d, 2H, J=15.9Hz), 7.00 (d, 2H, J=8.3 Hz), 7.25 (d, 2H, J=6.8 Hz), 7.33 (s, 2H), 7.57(d, 2H, J=15.7 Hz), 16.32 (s, 1H); ¹³C NMR: (DMSO) δ 55.6, 100.8, 110.5,111.7, 122.0, 122.7, 127.5, 140.2, 148.9, 150.9, 183.0.

3-Methyl-2,4-pentanedione (5). 2,4-Pentanedione (2, 6.3 ml, 60.2 mmol)and potassium carbonate (7.75 g, 56.1 mmol) were combined in acetone (12ml) and stirred for 15 min at room temperature. Methyl iodide (4, 4.6ml, 73.9 mmol) was added and the resulting mixture refluxed with acalcium chloride drying tube for 18 hr. An additional amount of methyliodide (1.5 ml, 24.1 mmol) was added and reflux was continued for 2 hr.The resulting mixture was filtered and the solvent evaporated to afforda liquid. The crude liquid was distilled to give 5.33 g (78%) of a clearliquid: bp 164-170° C.; ¹H NMR: δ enol form: 1.65 (s, 6H), 1.92 (s, 3H);keto form: 1.12 (d, 3H, J=7.0 Hz), 2.00 (s, 6H), 3.52 (q, 1H, J=7.0 Hz);¹³C NMR: δ 12.2, 12.6, 23.0, 28.4, 61.3, 104.4, 189.9, 204.5.

4-Methyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(6a). Boric anhydride (0.49 g, 7.0 mmol) was combined with3-methyl-2,4-pentanedione (5, 1.14 g, 10 mmol) and stirred for 24 hr atroom temperature under a nitrogen atmosphere. A solution of dry ethylacetate (10 ml), 4-hydroxy-3-methoxybenzaldehyde (1a, 3.04 g, 20.0 mmol)and tributyl borate (11.0 ml, 40.5 mmol) was added and the mixturestirred for 30 min at room temperature. Butylamine (0.2 ml, 2.0 mmol)was added dropwise over 40 min and stirring was continued for 24 hr atroom temperature. Hydrochloric acid (15 ml, 0.4 N) was warmed to 60° C.,added to the mixture and stirring was continued for 4 hr. The resultingmixture was filtered through celite and silica gel. The filtrate waswashed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to afford a solid. The crude solid wasrecrystallized three times from methanol to give 0.86 g (22%) of anorange solid: mp 180-183° C. [expected mp 180-183° C.]; ¹H NMR: δ 2.16(s, 3H), 3.94 (s, 6H), 5.83 (s, 2H), 6.94 (m, 4H), 7.04 (d, 2H, J=1.6Hz), 7.16 (d, 2H, J=8.0 Hz), 7.66 (d, 2H, J=15.5 Hz); ¹³C NMR: δ 11.5,13.1, 55.2, 55.6, 55.8, 105.6, 111.4, 111.6, 115.5, 117.7, 122.1, 123.2,123.5, 125.6, 126.6, 141.4, 143.7, 147.8, 149.1, 149.6, 182.1, 196.0;Anal. Calcd for C₂₂H₂₂O₆: C, 69.10; H, 5.80. Found: C, 69.19; H, 5.89.

4-Methyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (6b). Boric anhydride(0.49 g, 7.0 mmol) was combined with 3-methyl-2,4-pentanedione (5, 1.14g, 10.0 mmol) and stirred for 24 hr at room temperature under a nitrogenatmosphere. A solution of ethyl acetate (10 ml), benzaldehyde (1b, 2.05ml, 20.2 mmol) and tributyl borate (11.0 ml, 40.5 mmol) was added andthe mixture stirred for 15 min at room temperature. Butylamine (0.20 ml,2.0 mmol) was added dropwise over 30 min and stirring was continued for24 hr at room temperature. The resulting mixture was filtered to afforda solid. The crude solid was triturated with methanol to give 1.80 g(62%) of an orange solid: mp 154-157° C. [expected mp 154-157° C.]; ¹HNMR: δ 2.17 (s, 3H), 7.12 (d, 2H, J=15.5 Hz), 7.38 (m, 6H), 7.58 (m,4H), 7.74 (d, 2H, J=15.5 Hz); ¹³C NMR: δ 12.1, 106.2, 120.8, 182.1,128.8, 129.9, 135.4, 141.3, 182.4. Anal. Calcd for C₂₀H₁₈O₂: C, 82.73;H, 6.25. Found: C, 82.69; H, 6.36.

3-Benzylidene-2,4-pentanedione (7). 2,4-Pentanedione (2, 4.10 ml, 39.2mmol) and benzaldehyde (1b, 4.06 ml, 40.0 mmol) were stirred in benzene(10 ml). Piperdine (3 drops) and glacial acetic acid (6 drops) wereadded and the mixture refluxed with a Dean-Stark water trap for 3 hr.The resulting mixture was cooled to room temperature, extracted intoethyl ether, washed with hydrochloric acid (1 N), saturated sodiumbicarbonate, hydrochloric acid (1 N) and twice with water. The organiclayer was dried over magnesium sulfate, filtered and evaporated toafford an oil. The crude oil was distilled bulb to bulb to give 7.03 g(95%) of a yellow oil; [expected mp 165-167° C.]; ¹H NMR: δ 2.24 (s,3H), 2.38 (s, 3H), 7.35 (s, 5H), 7.45 (s, 1H); ¹³C NMR: δ 26.5, 31.6,128.9, 129.6, 130.5, 132.8, 139.6, 142.7, 196.2, 205.3.

3-Benzyl-2,4-pentanedione (8). 3-Benzylidene-2,4-pentanedione (7, 6.50g, 34.5 mmol) and palladium on activated carbon (0.25 g, 10%) werecombined in ethyl acetate (50 ml). The mixture was placed under ahydrogen atmosphere (60 psi) on a Parr apparatus for 4 hr at roomtemperature. The resulting mixture was filtered through celite and thesolvent evaporated to afford an oil. The crude oil was distilled bulb tobulb to give 6.52 g (99%) of a clear oil; ¹H NMR: δ enol form: 2.02 (s,6H), 3.62 (s, 2H), 7.24 (m, 5H); keto form: 2.07 (s, 6H), 3.11 (d, 2H,J=7.4 Hz), 4.01 (t, 1H, J=7.7 Hz), 7.24 (m, 5H); ¹³C NMR: δ 22.9, 29.4,32.5, 33.8, 69.2, 107.9, 125.9, 126.3, 127.0, 128.1, 128.2, 137.7,139.3, 191.4, 202.9.

4-Benzyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(9a). Boric anhydride (0.49 g, 7.0 mmol) was combined with3-benzyl-2,4-pentanedione (8, 1.90 g, 10 mmol) and stirred for 18 hr atroom temperature under a nitrogen atmosphere. A solution of dry ethylacetate (15 ml), 4-hydroxy-3-methoxybenzaldehyde (1a, 3.04 ml, 20.0mmol) and tributyl borate (11.0 ml, 40.5 mmol) was added and the mixturestirred for 30 min at room temperature. Butylamine (0.2 ml, 2.0 mmol)was added dropwise over 40 min and stirring was continued for 48 hr atroom temperature. Hydrochloric acid (I5 ml, 0.5 N) was warmed to 60° C.,added to the mixture and stirring was continued for 1 hr. The resultingmixture was extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a solid. The crude solid was recrystallized three times frommethanol to give 2.73 g (59%) of a orange-yellow solid: mp 144-146° C.[expected mp 139-141° C.]; ¹H NMR: (DMSO) δ 3.81 (s, 6H), 4.11 (s, 2H),6.78 (d, 2H, J=8.1 Hz), 7.20 (m, 11H), 7.58 (d, 2H, J=15.1 Hz), 9.66 (s,2H); ¹³C NMR: (DMSO) δ 30.2, 33.7, 55.6, 55.7, 63.0, 109.9, 111.3,115.5, 117.9, 122.4, 123.2, 123.7, 125.5, 125.7, 126.0, 126.5, 127.7,128.1, 128.3, 128.7, 139.1, 141.7, 142.3, 144.1, 147.8, 149.2, 149.7,183.0, 194.0; Exact mass calcd for C₂₈H₂₆O₆: 458.1729, observed (M+H)459.1798.

4-Benzyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (9b). Boric anhydride(0.49 g, 7.0 mmol) was combined with 3-benzyl-2,4-pentanedione (8, 1.90g, 10.0 mmol) and stirred for 48 hr at room temperature under a nitrogenatmosphere. A solution of dry ethyl acetate (10 ml), benzaldehyde (1b,2.05 ml, 20.2 mmol) and tributyl borate (11.0 ml, 40.5 mmol) was addedand the mixture stirred for 15 min at room temperature. Butylamine (0.20ml, 2.0 mmol) was added dropwise over 30 min and stirring was continuedfor 18 hr at room temperature. Hydrochloric acid (15 ml, 0.4 N) waswarmed to 60° C., added to the mixture and stirring was continued for 1hr. The resulting mixture was filtered to afford a solid. The crudesolid was triturated with methanol to give 2.30 g (63%) of a yellowsolid: mp 162-164° C. [expected mp 156-158° C.]; ¹H NMR: δ 3.99 (s, 2H),6.99 (d, 2H, J=15.6 Hz), 7.34 (m, 15H), 7.77 (d, 2H, J=15.2 Hz); ¹³CNMR: δ 31.8, 109.3, 120.8, 126.5, 127.8, 128.1, 128.8, 130.0, 135.3,140.5, 141.9, 183.6.

4,4-Dimethyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (11 b).1,7-Diphenyl-1,6-heptadiene-3,5-dione (3b, 0.30 g, 1.1 mmol) was stirredin dichloromethane (10 ml) for 5 min at room temperature. A solution ofsodium hydroxide (0.10 g, 2.5 mmol), tetrabutylammonium chloride (0.42g, 1.5 mmol) and water (3 ml) was added and the mixture stirred for 10min at room temperature. Methyl iodide (4, 0.21 ml, 3.4 mmol) was addedand the mixture stirred for 1 hr at 40° C. The mixture was cooled toroom temperature, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford an oil. The crudeoil was distilled bulb to bulb to give 0.25 g (76%) of a yellow oil; ¹HNMR: δ 1.46 (s, 6H), 6.77 (d, 2H, J=15.7 Hz), 7.33 (m, 6H), 7.49 (m,4H), 7.72 (d, 2H, J=15.6 Hz); ¹³C NMR: δ 21.1, 60.9, 121.4, 128.5,128.7, 130.6, 134.1, 144.1, 197.9; Anal. Calcd for C₂₀H₁₈O₂: C, 82.86;H, 6.62. Found: C, 82.54; H, 6.72.

4,4-Dibenzyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (12b).1,7-Diphenyl-1,6-heptadiene-3,5-dione (3b, 0.25 g, 0.9 mmol) was stirredin dichloromethane (4 ml) for 5 min at room temperature. A solution ofsodium hydroxide (80.0 mg, 2.0 mmol), tetrabutylammonium chloride (0.29g, 1.0 mmol) and water (2 ml) was added and the mixture stirred for 10min at room temperature. Benzyl bromide (10, 0.22 ml, 1.8 mmol) wasadded and the mixture stirred for 1 hr at 40° C. The resulting mixturewas cooled to room temperature, washed with saturated sodium chloride,dried over magnesium sulfate, filtered and evaporated to afford a solid.The crude solid was chromatographed on silica gel with ethylacetate/hexane to give a solid. The solid was recrystallized frommethanol to give 0.25 g (61%) of a white solid: mp 182-183° C. [expectedmp 181° C.]; ¹H NMR: δ 3.39 (s, 4H), 6.70 (d, 2H, J=15.5 Hz), 7.09-7.44(m, 20H), 7.73 (d, 2H, J=15.5 Hz); ¹³C NMR: δ 37.7, 70.3, 123.1, 126.7,128.1, 128.6, 128.8, 130.3, 130.7, 134.2, 136.3, 142.7, 196.8.

1,7-Bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (13a).1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (3a, 0.55 g,1.5 mmol) and palladium on activated carbon (0.25 g, 5%) were combinedin ethyl acetate (30 ml). The mixture was placed under a hydrogenatmosphere (60 psi) on a Parr apparatus for 4 hr at room temperature.The resulting mixture was filtered through celite and the solventevaporated to afford a solid. The crude solid was recrystallized fromethyl acetate/hexane to give 0.30 g (54%) of white crystals: mp 92-94°C. [expected mp 92-93° C.]; ¹H NMR: δ 2.53 (t, 4H, J=7.9 Hz), 2.83 (m,4H), 3.84 (s, 6H), 5.40 (s, 1H), 5.48 (s, 2H), 6.64 (m, 4H), 6.81 (d,2H, J=8.3 Hz), 15.44 (s, 1H); ¹³C NMR: δ 29.2, 31.3, 40.4, 45.5, 55.9,99.7, 110.9, 111.0, 114.3, 120.7, 132.4, 143.9, 146.3, 193.0.

1,7-Diphenylheptane-3,5-dione (13b).1,7-Diphenyl-1,6-heptadiene-3,5-dione (3b, 0.56 g, 2.0 mmol) andpalladium on activated carbon (0.25 g, 5%) were combined in ethylacetate (40 ml). The mixture was placed under a hydrogen atmosphere (60psi) on a Parr apparatus for 4 hr at room temperature. The resultingmixture was filtered through celite and the solvent evaporated to affordan oil. The crude oil was purified by preparative thin layerchromatography with ethyl acetate/hexane to give 0.40 g (70%) of anorange-yellow oil; ¹H NMR: δ enol form: 2.53 (m, 4H), 2.83 (m, 4H), 5.47(s, 1H), 7.30 (m, 10H); keto form: 2.53 (m, 4H), 2.83 (m, 4H), 3.54 (s,2H), 7.30 (m, 10H); ¹³C NMR: δ 29.4, 31.5, 39.9, 45.0, 99.5, 126.1,128.3, 128.5, 140.5, 170.4, 172.0, 192.8.

4-Methyl-1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (14a).4-Methyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(6a, 0.20 g, 0.5 mmol) and palladium on activated carbon (0.25 g, 10%)were combined in ethyl acetate (100 ml). The mixture was placed under ahydrogen atmosphere (60 psi) on a Parr apparatus for 2 hr at roomtemperature. The resulting mixture was filtered through celite and thesolvent evaporated to afford an oil. The crude oil was twicechromatographed on silica gel with ethyl acetate/hexane to give asemi-solid. The crude semi-solid was distilled bulb to bulb to give 80mg (38%) of a pale yellow oil; ¹H NMR: δ enol form: 1.69 (s, 3H), 2.72(m, 8H), 3.83 (s, 6H), 5.48 (s, 2H), 6.69 (m, 4H), 6.79 (d, 2H, J=7.5Hz); keto for: 1.23 (d, 3H, J=7.2 Hz), 2.72 (m, 8H), 3.57 (q, 1H, J=7.0Hz), 3.83 (s, 6H), 5.48 (s, 2H), 6.69 (m, 4H), 6.79 (d, 2H, J=7.5 Hz);¹³C NMR: δ 12.5, 29.2, 43.3, 55.9, 61.4, 111.0, 114.2, 120.7, 132.4,143.9, 146.3, 206.1; Exact mass calcd for C₂₂H₂₆O₆: 386.1729, observed(M+H) 387.1783.

4-Methyl-1,7-diphenylheptane-3,5-dione (14b).4-Methyl-1,7-diphenyl-1,6-hetpadiene-3,5-dione (6b, 0.96 g, 3.3 mmol)and palladium on activated carbon (0.25 g, 10%) were combined in ethylacetate (50 ml). The mixture was placed under a hydrogen atmosphere (60psi) on a Parr apparatus for 2 hr at room temperature. The resultingmixture was filtered through celite and the filtrate was washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford an oil. The crude oil was twice chromatographed onsilica gel with ethyl acetate/hexane to give an oil. The oil wasdistilled bulb to bulb to give 0.71 g (73%) of a clear oil; ¹H NMR: δenol form: 1.66 (s, 3H), 2.73 (m, 8H), 7.17 (m, 10H); keto form: 1.20(d, 3H, J=7.2 Hz), 2.73 (m, 8H), 3.55 (q, 1H, J=7.0 Hz), 7.17 (m, 10H);¹³C NMR: δ 12.3, 29.3, 31.0, 37.6, 42.8, 60.9, 104.2, 125.9, 128.1,128.2, 140.4, 140.8, 174.6, 191.5, 205.6; Exact mass calcd for C₂₀H₂₂O₂:294.1620, observed (M+H) 295.1693.

4-Benzyl-1,7-bis(4-hydroxy-3-methoxyphenyl)heptane-3,5-dione (15a).4-Benzyl-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione(9a, 0.25 g, 0.5 mmol) and palladium on activated carbon (0.20 g, 10%)were combined in ethyl acetate (45 ml). The mixture was placed under ahydrogen atmosphere (60 psi) on a Parr apparatus for 2 hr at roomtemperature. The resulting mixture was filtered through celite and thefiltrate was washed with saturated sodium chloride, dried over magnesiumsulfate, filtered and evaporated to afford an oil. The crude oil wastwice chromatographed on silica gel with ethyl acetate/hexane to give0.12 g (48%) of a pale yellow oil; ¹H NMR: δ enol form: 2.66 (m, 8H),3.53 (s, 2H), 3.77 (s, 6H), 5.55 (s, 2H), 6.56 (m, 4H), 6.77 (d, 2H,J=7.6 Hz), 7.06 (m, 6H), 7.20 (m, 2H); keto form: 2.66 (m, 8H), 3.08 (d,2H, J=7.3 Hz), 3.82 (s, 6H), 3.92 (t, 1H, J=8.3 Hz), 5.55 (s, 2H), 6.56(m, 4H), 6.77 (d, 2H, J=7.6 Hz), 7.06 (m, 6H), 7.20 (m, 2H); ¹³C NMR: δ29.0, 31.1, 31.8, 34.3, 37.7, 44.6, 55.8, 69.2, 111.0, 114.2, 120.7,120.8, 126.6, 127.4, 128.5, 128.6, 132.3, 132.6, 137.9, 143.8, 146.3,193.4, 204.5; Exact mass calcd for C₂₈H₃₀O₆: 462.2042, observed (M+H)463.2073.

4-Benzyl-1,7-diphenylheptane-3,5-dione (15b).4-Benzyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (9b, 0.26 g, 0.7 mmol)and palladium on activated carbon (0.25 g, 10%) were combined in ethylacetate (50 ml). The mixture was placed under a hydrogen atmosphere (60psi) on a Parr apparatus for 2 hr at room temperature. The resultingmixture was filtered through celite and the filtrate was washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford an oil. Hexane was added to the crude oil and theresulting precipitate was filtered. The crude solid was recrystallizedtwice from hexane to give 0.18 g (69%) of white needles: mp 74-75° C.;¹H NMR: δ enol form: 2.61 (m, 10H), 7.14 (m, 15H); keto form: 2.61 (m,8H), 3.07 (d, 2H, J=7.2 Hz), 3.90 (t, 1H, J=7.6 Hz), 7.14 (m, 15H); ¹³CNMR: δ 29.3, 34.3, 44.3, 69.2, 126.1, 126.7, 128.3, 128.4, 128.6, 128.7,137.9, 140.4, 204.3; Exact mass calcd for C₂₆H₂₆O₂: 370.1933, observed(M+H) 371.2014.

4,4-Dimethyl-1,7-diphenylheptane-3,5-dione (16b).4,4-Dimethyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (11b, 0.15 g, 0.5mmol) and palladium on activated carbon (0.20 g, 10%) were combined inethyl acetate (25 ml). The mixture was placed under a hydrogenatmosphere (60 psi) on a Parr apparatus for 1 hr at room temperature.The resulting mixture was filtered through celite and the filtrate waswashed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to afford an oil. The crude oil waschromatographed on silica gel with ethyl acetate/hexane to give 0.12 g(80%) of a pale yellow oil; ¹H NMR: δ 1.25 (s, 6H), 2.60 (t, 4H, J=7.4Hz), 2.80 (t, 4H, J=7.0 Hz), 7.18 (m, 10H); ¹³C NMR: δ 21.1, 29.8, 40.2,62.4, 126.1, 128.3, 140.7, 208.4; Exact mass calcd for C₂₁H₂₄O₂:308.1776, observed (M+H) 309.1843.

4,4-Dibenzyl-1,7-diphenylheptane-3,5-dione (17b).4,4-Dibenzyl-1,7-diphenyl-1,6-heptadiene-3,5-dione (12b, 70 mg, 0.2mmol) and palladium on activated carbon (0.10 g, 10%) were combined inethyl acetate (25 ml). The mixture was placed under a hydrogenatmosphere (60 psi) on a Parr apparatus for 5 hr at room temperature.The resulting mixture was filtered through celite and the filtrate waswashed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to afford a solid. The crude solid waschromatographed on silica gel with ethyl acetate/hexane to give a solid.The solid was recrystallized from hexane to give 60 mg (86%) of a whitesolid: mp 101-102° C. [expected mp 100.5-101.5° C.]; ¹H NMR: δ 2.56 (t,4H, J=7.4 Hz), 2.74 (t, 4H, J=6.8 Hz), 3.29 (s, 4H), 7.04 (m, 20H); ¹³CNMR: δ 29.6, 37.3, 42.5, 71.1, 126.1, 126.8, 128.4, 129.6, 136.0, 140.6,207.8; Anal. Calcd for C₃₃H₃₂O₂: C, 86.05; H, 7.00. Found: C, 86.28; H,7.11.

1,5-Bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one (20a).1,5-Bis(4-methoxymethoxy-3-methoxyphenyl)-1,4-pentadien-3-one (20j, 0.41g, 10.0 mmol) was stirred in methanol (50 ml) for 15 min at 50° C.Concentrated hydrochloric acid (1 drop) was added and the solutionstirred for 3 hr at 50° C. The methanol was evaporated and the resultingresidue extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a semi-solid. The crude semi-solid was purified by preparativethin layer chromatography with ethyl acetate/hexane to give 0.31 g (96%)of a yellow solid: mp 84-86° C. [expected mp 82-83° C.]; ¹H NMR: δ 3.89(s, 6H), 6.87 (d, 2H, J=8.4 Hz), 6.88 (d, 2H, J=15.9 Hz), 7.10 (m, 4H),7.62 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 56.1, 109.8, 114.8, 123.3, 123.4,127.5, 143.0, 146.8, 148.1, 188.6.

1,5-Diphenyl-1,4-pentadien-3-one (20b). Benzaldehyde (1b, 2.54 ml, 25.0mmol) and acetone (19, 0.90 ml, 12.2 mmol) were combined in ethanol (20ml) and stirred for 15 min at room temperature. A solution of sodiumhydroxide (2.50 g, 62.5 mmol) and water (25 ml) was added and thesolution stirred for 3 hr at room temperature. The resulting precipitatewas filtered and recrystallized from ethanol to afford 2.35 g (82%) ofyellow crystals: mp 110-112° C. [expected mp 112-114° C.]; ¹H NMR: δ7.07 (d, 2H, J=15.9 Hz), 7.40 (m, 8H), 7.61 (m, 2H), 7.73 (d, 2H, J=15.9Hz); ¹³C NMR: δ 125.4, 128.3, 12.9, 130.4, 134.7, 143.2, 188.7.

1,5-Bis(2-methoxyphenyl)-1,4-pentadien-3-one (20c).2-Methoxybenzaldehyde (1c, 1.50 ml, 12.4 mmol) and acetone (19, 0.46 ml,6.2 mmol) were combined in ethanol (10 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (0.50 g, 12.5 mmol) andwater (10 ml) was added and the mixture stirred for 18 hr at roomtemperature. The resulting precipitate was filtered and recrystallizedfrom ethanol to give 1.56 g (85%) of a yellow solid: mp 123-124° C.[expected mp 124° C.]; ¹H NMR: δ 3.87 (s, 6H), 6.91 (m, 4H), 7.15 (d,2H, J=16.1 Hz), 7.33 (dt, 2H, J=7.2, 1.4 Hz), 7.59 (d, 2H, J=7.5 Hz),8.06 (d, 2H, J=16.3 Hz); ¹³C NMR: δ 55.4, 111.1, 120.6, 123.8, 126.1,128.5, 131.4, 138.0, 158.4, 189.6.

1,5-Bis(2,3-dimethoxyphenyl)-1,4-pentadien-3-one (20d).2,3-Dimethoxybenzaldehyde (1d, 4.50 g, 27.1 mmol) and acetone (19, 1.00ml, 13.5 mmol) were combined in ethanol (25 ml) and stirred for 15 minat room temperature. A solution of sodium hydroxide (2.20 g, 55.0 mmol)and water (25 ml) was added and the mixture stirred for 6 hr at roomtemperature. The resulting precipitate was filtered and recrystallizedfrom ethanol to give 4.15 g (87%) of a yellow solid: mp 106-108° C.[expected mp 108° C.]; ¹H NMR: δ 3.89 (s, 6H), 3.90 (s, 6H), 6.96 (d,2H, J=8.1 Hz), 7.09 (t, 2H, J=8.1 Hz), 7.16 (d, 2H, J=16.3 Hz), 7.25 (d,2H, J=7.4 Hz), 8.05 (d, 2H, J=16.1 Hz); ¹³C NMR: δ 55.9, 61.3, 114.1,119.3, 124.1, 126.8, 129.0, 137.8, 148.7, 153.0, 189.5.

1,5-Bis(4-methoxyphenyl)-1,4-pentadien-3-one (20e).4-Methoxybenzaldehyde (1e, 1.50 ml, 12.3 mmol) and acetone (19, 0.45 ml,6.2 mmol) were combined in ethanol (20 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (2.53 g, 63.3 mmol) andwater (25 ml) was added and the mixture stirred for 3 hr at roomtemperature. The resulting precipitate was filtered and recrystallizedfrom ethanol to give 1.00 g (55%) of a yellow solid: mp 128-130° C.[expected mp 133-134° C.]; ¹H NMR: δ 3.82 (s, 6H), 6.90 (d, 4H, J=8.5Hz), 6.93 (d, 2H, J=15.9 Hz), 7.54 (d, 4H, J=8.5 Hz), 7.68 (d, 2H,J=15.9 Hz); ¹³C NMR: δ 55.4, 114.4, 123.5, 127.6, 129.9, 142.5, 161.4,188.6.

1,5-Bis(4-hydroxyphenyl)-1,4-pentadien-3-one (20f).4-Hydroxybenzaldehyde (1f, 2.00 g, 16.4 mmol) and acetone (19, 0.61 ml,8.3 mmol) were combined in ethanol (30 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (1.00 g, 25.0 mmol) andwater (30 ml) was added and the mixture stirred for 4 hr at roomtemperature. The resulting mixture was extracted into ethyl acetate,washed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to afford a solid. The crude solid wasrecrystallized from ethyl acetate/hexane to give 0.85 g (39%) of ayellow solid: mp 235-237° C. [expected mp 238-239° C.]; ¹H NMR: (DMSO) δ6.82 (d, 4H, J=8.5 Hz), 7.08 (d, 2H, J=16.1 Hz), 7.61 (d, 4H, J=8.5 Hz),7.64 (d, 2H, J=15.7 Hz), 10.08 (s, 2H); ¹³C NMR: (DMSO) δ 115.8, 122.6,125.7, 130.3, 142.3, 159.7, 187.9.

1,5-Bis(4-dimethylaminophenyl)-1,4-pentadien-3-one (20g).4-Dimethylaminobenzaldehyde (1g, 1.00 g, 6.7 mmol) and acetone (19, 0.24ml, 3.2 mmol) were combined in ethanol (10 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (0.40 g, 10 mmol) andwater (10 ml) was added and the mixture stirred for 18 hr at roomtemperature. The resulting mixture was extracted into ethyl acetate,washed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to afford a solid. The crude solid wasrecrystallized from ethanol to give 0.53 g (51%) of an orange solid: mp179-181° C. [expected mp 174-176° C.]; ¹H NMR: δ 3.01 (s, 12H), 6.69 (d,4H, J=8.7 Hz), 6.87 (d, 2H, J=15.7 Hz), 7.50 (d, 4H, J=8.7 Hz), 7.67 (d,2H, J=15.7 Hz); ¹³C NMR: δ 40.2, 98.9, 111.8, 121.2, 122.9, 129.9,142.8, 151.6.

1,5-Bis(3,4-dimethoxyphenyl)-1,4-pentadien-3-one (20i).3,4-Dimethoxybenzaldehyde (1i, 2.25 g, 13.5 mmol) and acetone (19, 0.50ml, 6.8 mmol) were combined in ethanol (15 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (1.10 g, 27.5 mmol) andwater (10 ml) was added and the mixture stirred for 2 hr at roomtemperature. The resulting precipitate was filtered and recrystallizedfrom ethanol to give 1.80 g (75%) of a yellow solid: mp 72-75° C.[expected mp 68-70° C.]; ¹H NMR: δ 3.92 (s, 6H), 3.94 (s, 6H), 6.89 (d,2H, J=8.3 Hz), 6.96 (d, 2H, J=15.9 Hz), 7.14 (s, 2H), 7.20 (d, 2H, J=8.1Hz), 7.69 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 55.9, 109.9, 111.0, 122.9,123.5, 127.7, 142.8, 149.1, 151.2, 188.4.

1,5-Bis(4-methoxymethyloxy-3-methoxyphenyl)-1,4-pentadien-3-one (20j).4-Methoxymethyloxy-3-methoxybenzaldehyde (1j, 1.95 g, 10.0 mmol) andacetone (19, 0.37 ml, 5.0 mmol) were combined in ethanol (25 ml) andstirred for 15 min at room temperature. A solution of sodium hydroxide(0.65 g, 16.3 mmol) and water (25 ml) was added and the solution stirredfor 18 hr at room temperature. The resulting mixture was extracted intodichloromethane, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford an oil. The crudeoil was chromatographed on silica gel with ethyl acetate/hexane to give1.40 g (67%) of a yellow solid: mp 81-82° C.; ¹H NMR: δ 3.53 (s, 6H),3.95 (s, 6H), 5.29 (s, 4H), 6.97 (d, 2H, J=15.9 Hz), 7.17 (m, 6H), 7.69(d, 2H, J=15.9 Hz); ¹³C NMR: δ 56.0, 56.4, 95.2, 110.8, 115.9, 122.5,124.0, 124.6, 129.2, 142.8, 148.7, 149.8, 188.5.

1,5-Bis(3-methoxyphenyl)-1,4-pentadien-3-one (20k).3-Methoxybenzaldehyde (1k, 3.09 ml, 25.4 mmol) and acetone (19, 0.94 ml,12.7 mmol) were combined in ethanol (20 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (1.50 g, 37.5 mmol) andwater (20 ml) was added and the mixture stirred for 18 hr at roomtemperature. The resulting mixture was extracted into ethyl acetate,washed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to afford an oil. The crude oil waschromatographed on silica gel with ethyl acetate/hexane to yield asolid. The solid was recrystallized from ethanol to give 2.06 g (62%) ofa yellow solid: mp 64-65° C. [expected mp 52-54° C.]; ¹H NMR: δ 3.83 (s,6H), 6.94 (dd, 2H, J=8.1, 2.4 Hz), 7.04 (d, 2H, J=15.9 Hz), 7.15 (m,4H), 7.32 (t, 2H, J=8.0 Hz), 7.68 (d, 2H, J=16.1 Hz); ¹³C NMR: δ 55.2,113.2, 116.2, 120.9, 125.5, 129.8, 136.0, 143.0, 159.8, 188.6.

1,5-Bis(2,6-dimethoxyphenyl)-1,4-pentadien-3-one (20l).2,6-Dimethoxybenzaldehyde (20l, 1.00 g, 6.0 mmol) and acetone (19, 0.44ml, 3.0 mmol) were combined in ethanol (10 ml) and stirred for 10 min atroom temperature. A solution of sodium hydroxide (0.72 g, 9.0 mmol) andwater (15 ml) was added and the mixture stirred for 18 hr at roomtemperature. The resulting precipitate was filtered and recrystallizedfrom ethanol to give 0.66 g (63%) of a yellow solid: mp 152-154° C.[expected mp 152-154° C.]; ¹H NMR: δ 3.90 (s, 12H), 6.57 (d, 4H, J=8.5Hz), 7.26 (t, 2H, J=8.5 Hz), 7.59 (d, 2H, J=16.3 Hz), 8.17 (d, 2H,J=16.3 Hz); ¹³C NMR: δ 55.8, 103.7, 113.1, 129.0, 130.9, 133.3, 160.0,192.4.

1,5-Bis(2,5-dimethoxyphenyl)-1,4-pentadien-3-one (20m).2,5-Dimethoxybenzaldehyde (1m, 2.00 g, 12.0 mmol) and acetone (19, 0.44ml, 6.0 mmol) were combined in ethanol (15 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (0.72 g, 18.0 mmol) andwater (15 ml) was added and the mixture stirred for 18 hr at roomtemperature. The resulting precipitate was filtered and recrystallizedfrom ethanol to give 1.46 g (69%) of a yellow solid: mp 105-106° C.[expected mp 105-106° C.]; ¹H NMR: δ 3.79 (s, 6H), 3.85 (s, 6H), 6.88(m, 4H), 7.11 (d, 2H, J=2.8 Hz), 7.12 (d, 2H, J=16.1 Hz), 8.01 (d, 2H,J=16.1 Hz); ¹³C NMR: δ 55.8, 56.1, 112.4, 113.1, 117.1, 124.5, 126.3,137.9, 153.0, 153.4, 189.6.

1,5-Bis(2,4-dimethoxyphenyl)-1,4-pentadien-3-one (20n).2,4-Dimethoxybenzaldehyde (1n, 2.00 g, 12.0 mmol) and acetone (19, 0.44ml, 6.0 mmol) were combined in ethanol (15 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (0.72 g, 18.0 mmol) andwater (15 ml) was added and the mixture stirred for 18 hr at roomtemperature. The resulting precipitate was filtered and recrystallizedfrom ethanol to give 1.71 g (81%) of a yellow solid: mp 138-140° C.[expected mp 138-139° C.]; ¹H NMR: δ 3.81 (s, 6H), 3.85 (s, 6H), 6.43(d, 2H, J=2.2 Hz), 6.48 (dd, 2H, J=8.5, 2.2 Hz), 7.04 (d, 2H, J=16.1Hz), 7.52 (d, 2H, J=8.5 Hz), 7.96 (d, 2H, J=16.1 Hz); ¹³C NMR: δ 55.4,55.5, 98.3, 105.3, 117.1, 124.1, 130.0, 137.6, 159.9, 162.6, 189.7.

1,5-Bis(3,5-dimethoxyphenyl)-1,4-pentadien-3-one (20o).3,5-Dimethoxybenzaldehyde (1o, 2.00 g, 12.0 mmol) and acetone (19, 0.44ml, 6.0 mmol) were combined in ethanol (15 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (0.72 g, 18.0 mmol) andwater (15 ml) was added and the mixture stirred for 18 hr at roomtemperature. The resulting precipitate was filtered and recrystallizedfrom ethanol to give 1.32 g (63%) of a yellow solid: mp 126-128° C.[expected mp 124.5-125.5° C.]; ¹H NMR: δ 3.80 (s, 12H), 6.49 (s, 2H),6.73 (d, 4H, J=2.0 Hz), 7.00 (d, 2H, J=15.9 Hz), 7.62 (d, 2H, J=15.7Hz); ¹³C NMR: δ 55.4, 102.7, 106.2, 125.7, 136.8, 143.2, 160.9, 188.6.

1,5-Bis(3-hydroxyphenyl)-1,4-pentadien-3-one (20p).3-Hydroxybenzaldehyde (1p, 2.07 g, 17.0) and acetone (19, 0.62 ml, 8.4mmol) were combined in ethanol (15 ml) and stirred for 15 min at roomtemperature. A solution of sodium hydroxide (1.00 g, 25.0 mmol) andwater (4 ml) was added and the solution stirred for 48 hr at roomtemperature. The resulting mixture was neutralized with hydrochloricacid (1 N), extracted with ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a solid. The crude solid was recrystallized from ethyl acetate togive 0.42 g (19%) of a brown solid: mp 190-195° C. [expected mp 198-200°C.]; ¹H NMR: (DMSO) δ 6.83 (d, 2H, J=7.0 Hz), 7.22 (m, 8H), 7.68 (d, 2H,J=16.1 Hz), 9.63 (s, 2H); ¹³C NMR: (DMSO) δ 114.7, 117.5, 119.4, 125.4,129.7, 135.8, 142.7, 157.5, 168.2.

1,5-Bis(2-hydroxyphenyl)-1,4-pentadien-3-one (20q).2-Hydroxybenzaldehyde (1q, 1.81 ml, 17.0 mmol) and acetone (19, 0.62 ml,8.4 mmol) were combined in ethanol (15 ml) and stirred for 15 min atroom temperature. A solution of sodium hydroxide (1.00 g, 25.0 mmol) andwater (4 ml) was added and the solution stirred for 1 week at roomtemperature. The mixture was neutralized with hydrochloric acid (1 N)and the resulting precipitate filtered and recrystallized from ethylacetate/hexane to give 1.79 g (80%) of a yellow solid: mp 154-157° C.[expected mp 155° C.]; ¹H NMR: (DMSO) δ 6.89 (m, 4H), 7.27 (m, 4H), 7.68(d, 2H, J=7.4 Hz), 7.93 (d, 2H, J=16.1 Hz), 10.22 (s, 2H); ¹³C NMR:(DMSO) δ 116.1, 119.3, 121.3, 125.3, 128.5, 131.5, 137.6, 156.9, 188.5.

1,5-Bis(4-fluorophenyl)-1,4-pentadien-3-one (20r). 4-Fluorobenzaldehyde(1r, 0.75 ml, 7.0 mmol) and acetone (19, 0.26 ml, 3.5 mmol) werecombined in ethanol (30 ml) and stirred for 10 min at room temperature.A solution of sodium hydroxide (0.50 g, 12.5 mmol) and water (20 ml) wasadded and the mixture stirred for 18 hr at room temperature. Theresulting precipitate was filtered and recrystallized from ethanol toafford 0.82 g (86%) of a yellow solid: mp 150-152° C. [expected mp152-154° C.]; ¹H NMR: δ 6.97 (d, 2H, J=15.9 Hz), 7.09 (m, 4H), 7.58 (m,4H), 7.68 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 116.1, 1125.1, 130.2, 130.9,142.0, 164.0, 188.3.

1,5-Bis(3-fluorophenyl)-1,4-pentadien-3-one (20s). 3-Fluorobenzaldehyde(1s, 0.5 ml, 4.7 mmol) and acetone (19, 0.18 ml, 2.3 mmol) were combinedin ethanol (20 ml) and stirred for 10 min at room temperature. Asolution of sodium hydroxide (0.29 g, 7.3 mmol) and water (15 ml) wasadded and the mixture stirred for 18 hr at room temperature. Theresulting mixture was filtered and chromatographed on silica gel withethyl acetate/hexane to give a solid. The crude was recrystallized fromethanol to afford 0.26 g (42%) of yellow crystals: mp 96-97° C.[expected mp 96-97° C.]; ¹H NMR: δ 7.03 (d, 2H, J=16.1 Hz), 7.09 (m,2H), 7.35 (m, 6H), 7.67 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 114.4, 117.5,124.4, 126.3, 130.4, 136.9, 142.0, 162.9, 188.1; Anal. Calcd forC₁₇H₁₂OF₂: C, 75.55; H, 4.48. Found: C, 75.26; H, 4.65.

1,5-Bis(2-fluorophenyl)-1,4-pentadien-3-one (20t). 2-Fluorobenzaldehyde(1t, 0.5 ml, 4.7 mmol) and acetone (19, 0.18 ml, 2.4 mmol) were combinedin ethanol (20 ml) and stirred for 10 min at room temperature. Asolution of sodium hydroxide (0.29 g, 7.3 mmol) and water (15 ml) wasadded and the mixture stirred for 18 hr at room temperature. Theresulting mixture was filtered and chromatographed on silica gel withethyl acetate/hexane to give a solid. The crude solid was recrystallizedfrom ethanol to afford 0.27 g (41%) of yellow crystals: mp 68-72° C.[expected mp 68-70° C.]; ¹H NMR: δ 7.13 (m, 6H), 7.36 (m, 2H), 7.61 (dt,2H, J=7.6 Hz, 1.4 Hz), 7.84 (d, 2H, J=16.3 Hz); ¹³C NMR: δ 116.2, 122.8,124.4, 127.6, 129.3, 131.8, 135.9, 161.5, 188.7.

1,5-Bis(4-trifluoromethyl)-1,4-pentadien-3-one (20u).4-(Trifluoromethyl)benzaldehyde (1u, 0.50 ml, 3.7 mmol) and acetone (19,0.13 ml, 1.8 mmol) were combined in ethanol (15 ml) and stirred for 10min at room temperature. A solution of sodium hydroxide (0.22 g, 5.5mmol) and water (15 ml) was added and the mixture stirred for 18 hr atroom temperature. The resulting precipitate was filtered andrecrystallized from ethanol to afford 0.57 g (87%) of a yellow solid: mp151-154° C. [expected mp 156-157° C.]; ¹H NMR: δ 7.12 (d, 2H, J=15.9Hz), 7.69 (m, 8H), 7.73 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 121.6, 125.9,127.2, 128.5, 132.1, 138.0, 141.8, 187.9.

1,5-Bis(3-trifluoromethyl)-1,4-pentadien-3-one (20v).3-(Trifluoromethyl)benzaldehyde (1v, 0.5 ml, 3.7 mmol) and acetone (19,0.14 ml, 1.9 mmol) were combined in ethanol (15 ml) and stirred for 10min at room temperature. A solution of sodium hydroxide (0.23 g, 5.8mmol) and water (15 ml) was added and the mixture stirred for 18 hr atroom temperature. The resulting mixture was filtered and chromatographedon silica gel with ethyl acetate/hexane to give a solid. The crude solidwas recrystallized from ethanol to give 0.25 g (36%) of yellow crystals:mp 116-117° C. [expected mp 116-117° C.]; ¹H NMR: δ 7.12 (d, 2H, J=15.9Hz), 7.53 (t, 2H, J=7.6 Hz), 7.66 (d, 2H, J=8.0 Hz), 7.73 (d, 2H, J=7.2Hz), 7.82 (m, 4H); ¹³C NMR: δ 123.7, 124.7, 126.7, 126.8, 129.5, 131.5,131.6, 135.4, 141.8, 187.8; Anal. Calcd for C₁₉H₁₂OF₆: C, 61.63; H,3.27. Found: C, 61.82; H, 3.28.

1,5-Bis(2-trifluoromethylphenyl)-1,4-pentadien-3-one (20w).2-(Trifluoromethyl)-benzaldehyde (1w, 0.75 ml, 5.7 mmol) and acetone(19, 0.21 ml, 2.8 mmol) were combined in ethanol (20 ml) and stirred for10 min at room temperature. A solution of sodium hydroxide (0.34 g, 8.5mmol) and water (15 ml) was added and the mixture stirred for 18 hr atroom temperature. The resulting precipitate was filtered andrecrystallized from ethanol to afford 0.92 g (87%) of a yellow solid: mp131-133° C. [expected mp 131° C.]; ¹H NMR: δ 6.99 (d, 2H, J=15.9 Hz),7.48 (t, 2H, J=7.6 Hz), 7.56 (t, 2H, J=7.0 Hz), 7.70 (d, 2H, J=7.7 Hz),7.77 (d, 2H, J=7.6 Hz), 8.07 (d, 2H, J=15.7 Hz); ¹³C NMR: δ 123.9,126.2, 127.9, 128.8, 129.4, 129.7, 132.1, 133.7, 139.1, 188.0.

1,5-Bis(4-chlorophenyl)-1,4-pentadien-3-one (20x). 4-Chlorobenzaldehyde(1x, 1.00 g, 7.1 mmol) and acetone (19, 0.26 ml, 3.5 mmol) were combinedin ethanol (10 ml) and stirred for 15 min at room temperature. Asolution of sodium hydroxide (0.40 g, 10.0 mmol) and water (10 ml) wasadded and the mixture stirred for 3 hr at room temperature. Theresulting precipitate was filtered and recrystallized from ethyl acetateto give 0.75 g (70%) of yellow crystals: mp 187-189° C. [expected mp191-193° C.]; ¹H NMR: δ 7.00 (d, 2H, J=15.9 Hz), 7.37 (d, 4H, J=8.5 Hz),7.52 (d, 4H, J=8.5 Hz), 7.66 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 125.7,129.2, 129.5, 133.2, 136.4, 141.9, 188.1.

1,5-Bis(3-chlorophenyl)-1,4-pentadien-3-one (20y). 3-Chlorobenzaldehyde(1y, 2.00 ml, 17.7 mmol) and acetone (19, 0.65 ml, 8.8 mmol) werecombined in ethanol (20 ml) and stirred for 15 min at room temperature.A solution of sodium hydroxide (1.00 g, 25.0 mmol) and water (20 ml) wasadded and the mixture stirred for 2 hr at room temperature. Theresulting precipitate was filtered and recrystallized from ethyl acetateto give 2.41 g (90%) of a yellow solid: mp 125-127° C. [expected mp120-121° C.]; ¹H NMR: δ 7.03 (d, 2H, J=15.9 Hz), 7.33 (m, 4H), 7.45 (d,2H, J=6.6 Hz), 7.58 (m, 2H), 7.64 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 126.3,126.6, 127.9, 130.1, 130.3, 134.9, 136.5, 141.8, 188.0.

1,5-Bis(2-chlorophenyl)-1,4-pentadien-3-one (20z). 2-Chlorobenzaldehyde(1z, 2.00 ml, 17.8 mmol) and acetone (19, 0.65 ml, 8.8 mmol) werecombined in ethanol (20 ml) and stirred for 15 min at room temperature.A solution of sodium hydroxide (1.00 g, 25.0 mmol) and water (20 ml) wasadded and the mixture stirred for 18 hr at room temperature. Theresulting precipitate was filtered and recrystallized from ethyl acetateto give 1.80 g (67%) of a yellow solid: mp 119-121° C. [expected mp 110°C.]; ¹H NMR: δ 7.04 (d, 2H, J=16.1 Hz), 7.29 (m, 4H), 7.41 (m, 2H), 7.67(m, 2H), 8.11 (d, 2H, J=16.1 Hz); ¹³C NMR: δ 127.0, 127.5, 127.6, 130.1,131.1, 132.9, 135.3, 139.2, 188.4.

1,5-Bis(4-methylphenyl)-1,4-pentadien-3-one (20aa). 4-Methylbenzaldehyde(1aa, 1.50 ml, 12.7 mmol) and acetone (19, 0.47 ml, 6.4 mmol) werecombined in ethanol (10 ml) and stirred for 15 min at room temperature.A solution of sodium hydroxide (0.52 g, 13.0 mmol) and water (10 ml) wasadded and the mixture stirred for 1 hr at room temperature. Theresulting precipitate was filtered and recrystallized from ethanol togive 1.30 g (78%) of a yellow solid: mp 174-176° C. [expected mp171-172° C.]; ¹H NMR: δ 2.37 (s, 6H), 7.02 (d, 2H, J=15.9 Hz), 7.20 (d,4H, J=8.0 Hz), 7.50 (d, 4H, J=7.9 Hz), 7.70 (d, 2H, J=15.9 Hz); ¹³C NMR:δ 21.6, 124.6, 128.3, 129.6, 132.1, 140.8, 143.0, 188.9.

1,5-Bis(3-methylphenyl)-1,4-pentadien-3-one (20ab). 3-Methylbenzaldehyde(1ab, 3.00 ml, 25.4 mmol) and acetone (19, 0.94 ml, 12.7 mmol) werecombined in ethanol (20 ml) and stirred for 15 min at room temperature.A solution of sodium hydroxide (1.50 g, 37.5 mmol) and water (20 ml) wasadded and the mixture stirred for 18 hr at room temperature. Theresulting mixture was extracted into ethyl acetate, washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford a solid. The crude solid was recrystallized fromethanol to give 2.39 g (72%) of a yellow solid: mp 68-72° C. [expectedmp 68-72° C.]; ¹H NMR: δ 2.38 (s, 6H), 7.06 (d, 2H, J=15.9 Hz), 7.26 (m,4H), 7.40 (m, 4H), 7.70 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 21.3, 125.1,125.4, 128.6, 128.8, 131.1, 134.6, 138.4, 143.1, 188.6.

1,5-Bis(2-methylphenyl)-1,4-pentadien-3-one (20ac). 2-Methylbenzaldehyde(1ac, 1.45 ml, 12.5 mmol) and acetone (19, 0.46 ml, 6.3 mmol) werecombined in ethanol (20 ml) and stirred for 15 min at room temperature.A solution of sodium hydroxide (2.61 g, 65.3 mmol) and water (25 ml) wasadded and the mixture stirred for 3 hr at room temperature. Theresulting mixture was extracted into ethyl acetate, washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford a solid. The crude solid was recrystallized fromethanol to give 0.71 g (43%) of a yellow solid: mp 98-100° C. [expectedmp 94-96° C.]; ¹H NMR: δ 2.47 (s, 6H), 6.98 (d, 2H, J=15.9 Hz), 7.24 (m,6H), 7.64 (d, 2H, J=7.2 Hz), 8.03 (d, 2H, J=15.9 Hz); ¹³C NMR: δ 19.7,126.1, 126.2, 126.5, 129.9, 130.7, 133.6, 137.9, 140.5, 188.5.

1,5-Bis(4-carbmethoxyphenyl)-1,4-pentadien-3-one (20ae).4-Carbmethoxybenzaldehyde (1ae, 0.62 g, 3.8 mmol) and acetone (19, 0.14ml, 1.9 mmol) were combined in methanol (20 ml) and stirred under anitrogen atmosphere for 15 min at room temperature. A solution of sodiumhydroxide (0.15 g, 3.8 mmol) in water (5 ml) was added the mixturestirred for 18 hr at room temperature under a nitrogen atmosphere. Theresulting precipitate was filtered and recrystallized from xylene togive 0.29 g (44%) of a yellow solid: mp 206-210° C. [expected mp221-223° C.]; ¹H NMR: δ 3.92 (s, 6H), 7.12 (d, 2H, J=16.1 Hz), 7.65 (d,4H, J=8.1 Hz), 7.73 (d, 2H, J=15.9 Hz), 8.06 (d, 4H, J=8.0 Hz); ¹³C NMR:δ 52.3, 127.1, 128.1, 130.1, 131.6, 138.8, 142.1, 166.2, 188.0.

1,5-Bis(3,4-dihydroxyphenyl)-1,4-pentadien-3-one (20af).1,5-Bis(3,4-dimethoxyphenyl)-1,4-pentadien-3-one (20i, 0.56 g, 1.6 mmol)was dissolved in dichloromethane (10 ml) and stirred under a nitrogenatmosphere at −78° C. for 5 min. Boron tribromide (0.90 ml, 9.5 mmol)was added and stirring continued for 60 min at −78° C., 60 min at 0° C.and 60 min at room temperature. The mixture was poured into hydrochloricacid (30 ml, 1 N) and stirring was continued for 18 hr at roomtemperature. The resulting mixture was extracted into ethyl acetate,washed with water and saturated sodium chloride, dried over magnesiumsulfate, filtered and evaporated to afford a solid. The crude solid waschromatographed on silica gel with ethyl acetate/hexane to give 0.36 g(76%) of an orange solid: mp>250° C. [expected mp 221-223° C.]; ¹H NMR:(DMSO) δ 6.78 (d, 2H, J=8.1 Hz), 6.99 (d, 2H, J=15.9 Hz), 7.06 (d, 2H,J=7.9 Hz), 7.14 (s, 2H), 7.55 (d, 2H, J=15.7 Hz), 9.15 (s, 2H), 9.63 (s,2H); ¹³C NMR: (DMSO) δ 114.9, 115.6, 121.5, 122.5, 126.2, 142.5, 145.4,148.2, 187.6.

1,5-Bis(4-acetoxy-3-methoxyphenyl)-1,4-pentadien-3-one (20ag).1,5-Bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadien-3-one (20a, 0.32 g, 1.0mmol) was dissolved in acetic anhydride (21, 7.00 ml, 74.1 mmol) andstirred for 5 min at room temperature. Pyridine (0.70 ml, 8.7 mmol) wasadded and the mixture stirred for 30 min at 100° C. The resultingmixture was poured into water, extracted with ethyl acetate, washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford a solid. The crude solid was recrystallized fromtetrahydrofuran/hexane to give 0.36 g (88%) of a yellow solid: mp179-180° C. [expected mp 150° C.]; ¹H NMR: δ 2.31 (s, 6H), 3.87 (s, 6H),6.98 (d, 2H, J=15.9 Hz), 7.06 (d, 2H, J=8.1 Hz), 7.18 (m, 4H), 7.67 (d,2H, J=15.9 Hz); ¹³C NMR: δ 20.7, 56.0, 111.7, 121.4, 123.3, 125.5,133.7, 141.6, 142.6, 151.4, 168.5, 188.3; Exact mass calcd for C₂₃H₂₂O₇:410.1366, observed (M+H) 411.1444.

1,5-Bis(4-acetoxyphenyl)-1,4-pentadien-3-one (20ah).1,5-Bis(4-hydroxyphenyl)-1,4-pentadien-3-one (20f, 0.26 g, 1.0 mmol) wasdissolved in acetic anhydride (21, 7.00 ml, 74.1 mmol) and stirred for 5min at room temperature. Pyridine (0.70 ml, 8.7 mmol) was added and themixture stirred for 30 min at 100° C. The resulting mixture was pouredinto water, extracted with ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a solid. The crude solid was recrystallized fromtetrahydrofuran/hexane to give 0.28 g (82%) of a yellow solid: mp167-168° C. [expected mp 167-168° C.]; ¹H NMR: δ 2.30 (s, 6H), 7.00 (d,2H, J=15.9 Hz), 7.13 (d, 4H, J=8.3 Hz), 7.60 (d, 4H, J=8.2 Hz), 7.69 (d,2H, J=15.9 Hz); ¹³C NMR: δ 21.1, 122.1, 125.4, 129.4, 132.4, 142.1,152.2, 168.9, 188.3; Exact mass calcd for C₂₁H₁₈O₅: 350.1154, observed(M+H) 351.1232.

1,5-Bis(1-naphthyl)-1,4-pentadien-3-one (23). 1-Naphthaldehyde (22, 1.36ml, 10.0 mmol) and acetone (19, 0.37 ml, 5.0 mmol) were combined inethanol (10 ml) and stirred for 15 min at room temperature. A solutionof sodium hydroxide (0.40 g, 10.0 mmol) and water (10 ml) was added andthe mixture stirred for 18 hr at room temperature. The resulting mixturewas extracted into ethyl acetate, washed with saturated sodium chloride,filtered and evaporated to afford a solid. The crude solid wasrecrystallized from ethyl acetate to give 0.63 g (38%) of a yellowsolid: mp 132-133° C. [expected mp 128° C.]; ¹H NMR: δ 7.24 (d, 2H,J=15.7 Hz), 7.57 (m, 6H), 7.92 (m, 6H), 8.28 (d, 2H, J=8.0 Hz), 8.65 (d,2H, J=15.5 Hz); ¹³C NMR: δ 123.4, 125.1, 125.4, 126.2, 126.9, 128.1,128.7, 130.7, 131.7, 132.2, 133.7, 140.3, 188.5.

1,5-Bis(2-naphthyl)-1,4-pentadien-3-one (25). 2-Naphthaldehyde (24, 1.56g, 10.0 mmol) and acetone (19, 0.37 ml, 5.0 mmol) were combined inethanol (10 ml) and stirred for 15 min at room temperature. A solutionof sodium hydroxide (0.60 g, 15.0 mmol) and water (10 ml) was added andthe mixture stirred for 18 hr at room temperature. The resultingprecipitate was filtered and recrystallized from ethanol to give 1.16 g(69%) of a yellow solid: mp 244-246° C. [expected mp 243-244° C.]; ¹HNMR: δ 7.23 (d, 2H, J=15.9 Hz), 7.52 (m, 4H), 7.83 (m, 8H), 7.93 (d, 2H,J=15.9 Hz), 8.03 (s, 2H); ¹³C NMR: δ 123.6, 125.7, 126.7, 127.3, 127.8,128.6, 128.7, 130.5, 132.3, 133.3, 134.3, 143.1, 190.0.

1,5-Bis(4-pyridinium chloride)-1,4-pentadien-3-one (28).1,3-Acetonedicarboxylic acid (27, 1.05 g, 7.2 mmol) was dissolved inethanol (10 ml) and stirred for 15 min at room temperature.4-Pyridinecarboxaldehyde (26, 1.37 ml, 14.4 mmol) was added dropwise andthe mixture stirred for 2 hr at room temperature. Hydrochloric acid (5ml) was added and the mixture stirred for 1 hr at 80° C. The resultingprecipitate was filtered and recrystallized from water/acetone to give0.59 g (27%) of a yellow solid: mp 247-249° C. [expected mp 247-249°C.]; ¹H NMR: (D₂O) δ 7.54 (d, 2H, J=16.3 Hz), 7.78 (d, 2H, J=15.9 Hz),8.15 (d, 4H, J=6.6 Hz), 8.70 (d, 4H, J=6.6 Hz); ¹³C NMR: (D₂O) δ 128.2,136.2, 141.3, 144.2, 154.4, 193.0.

1,5-Bis(4-pyridyl)-1,4-pentadien-3-one (29). 1,5-Bis(4-pyridiniumchloride)-1,4-pentadien-3-one (28, 0.25 g, 0.8 mmol) and sodiumhydroxide (0.80 g, 20 mmol) were combined in water (20 ml) and stirredfor 15 min at room temperature. The resulting mixture was extracted withethyl acetate, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford a solid. The crudesolid was recrystallized from ethyl acetate/hexane to give 0.16 g (84%)of a yellow solid: mp 145-146° C. [expected mp 149° C.]; ¹H NMR: δ 7.17(d, 2H, J=15.9 Hz), 7.42 (d, 4H, J=5.6 Hz), 7.63 (d, 2H, J=15.9 Hz),8.67 (d, 4H, J=5.6 Hz); ¹³C NMR: δ 121.9, 128.6, 141.0, 141.6, 150.6,172.5.

1,5-Bis(3-pyridinium chloride)-1,4-pentadien-3-one (31).1,3-Acetonedicarboxylic acid (27, 1.05 g, 7.2 mmol) was dissolved inethanol (10 ml) and stirred for 15 min at room temperature.3-Pyridinecarboxaldehyde (30, 1.36 ml, 14.4 mmol) was added dropwise andthe mixture stirred for 2 hr at room temperature. Hydrochloric acid (5ml) was added and the mixture stirred for 1 hr at 80° C. The resultingmixture was filtered to afford a solid. The crude solid wasrecrystallized from water/acetone to give 1.57 g (71%) of a yellowsolid: mp>250° C. [expected mp>250° C.]; ¹H NMR: (D₂O) δ 7.40 (d, 2H,J=16.3 Hz), 7.78 (d, 2H, J=16.1 Hz), 8.02 (t, 2H, J=7.9 Hz), 8.70 (d,2H, J=5.6 Hz), 8.79 (d, 2H, J=7.9 Hz), 8.98 (s, 2H); ¹³C NMR: (D₂O) δ130.1, 132.8, 136.9, 139.9, 143.7, 144.3, 147.3, 193.0.

1,5-Bis(3-pyridyl)-1,4-pentadien-3-one (32). 1,5-Bis(3-pyridiniumchloride)-1,4-pentadien-3-one (31, 0.50 g, 1.6 mmol) and sodiumhydroxide (1.6 g, 40 mmol) were combined in water (40 ml) and stirredfor 15 min at room temperature. The resulting mixture was extracted withethyl acetate, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford a solid. The crudesolid was recrystallized from ethyl acetate/hexane to give 0.31 g (81%)of a yellow solid: mp 148-149° C. [expected mp 150° C.]; ¹H NMR: δ 7.11(d, 2H, J=16.1 Hz), 7.32 (m, 2H), 7.71 (d, 2H, J=15.9 Hz), 7.90 (d, 2H,J=6.2 Hz), 8.61 (d, 2H, J=4.6 Hz), 8.81 (s, 2H); ¹³C NMR: δ 123.7,126.7, 130.3, 134.4, 139.9, 149.9, 151.1, 198.6.

1,5-Bis(2-thienyl)-1,4-pentadien-3-one (34). 2-Thiophenecarboxaldehyde(33, 0.50 ml, 5.3 mmol) and acetone (19, 0.20 ml, 2.7 mmol) werecombined in ethanol (10 ml) and stirred for 10 min at room temperature.A solution of sodium hydroxide (0.30 g, 7.5 mmol) and water (10 ml) wasadded and the mixture stirred for 18 hr at room temperature. Theresulting precipitate was filtered and recrystallized from ethanol/waterto give 0.55 g (82%) of a yellow solid: mp 115-117° C. [expected mp115-117° C.]; ¹H NMR: δ 6.80 (d, 2H, J=15.5 Hz), 7.06 (dt, 2H, J=3.6,1.4 Hz), 7.31 (d, 2H, J=3.4 Hz), 7.39 (d, 2H, J=5.0 Hz), 7.82 (d, 2H,J=15.5 Hz); ¹³C NMR: δ 124.4, 128.2, 128.7, 131.7, 135.5, 140.2, 187.5.

1-(4-Hydroxy-3-methoxyphenyl)-1-buten-3-one (35a).1-(4-Methoxymethyloxy-3-methoxyphenyl)-1-buten-3-one (35j, 0.40 g, 1.7mmol) was dissolved in methanol (40 ml) and stirred for 15 min at 50° C.Hydrochloric acid (3 drops) was added and the mixture stirred for 18 hrat 65° C. The methanol was evaporated and the resulting residueextracted into ethyl acetate, washed with saturated sodium chloride,dried over magnesium sulfate, filtered and evaporated to afford a solid.The crude solid was chromatographed on silica gel with ethylacetate/hexane to give 0.24 g (74%) of an orange-yellow solid: mp120-122° C. [expected mp 128-129° C.]; ¹H NMR δ 2.34 (s, 3H), 3.91 (s,3H), 5.98 (s, 1H), 6.56 (d, 1H, J=16.1 Hz), 6.91 (d, 1H, J=8.2 Hz), 7.04(m, 2H), 7.43 (d, 1H, J=16.3 Hz); ¹³C NMR: δ 27.3, 56.0, 109.3, 114.8,123.4, 124.9, 126.9, 143.6, 146.8, 148.2, 198.2.

1-(4-Methoxyphenyl)-1-buten-3-one (35e). 4-Methoxybenzaldehyde (1e, 0.63ml, 5.2 mmol) and acetone (19, 4.00 ml, 54.0 mmol) were combined inethanol (4 ml) and stirred for 15 min at room temperature. A solution ofsodium hydroxide (0.40 g, 10.0 mmol) and water (4 ml) was added dropwiseand the mixture stirred for 1 hr at room temperature. The resultingmixture was extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford a solid. The crude solid was recrystallized from ether/hexane togive 0.57 g (62%) of a yellow solid: mp 71-73° C. [expected mp 68° C.];¹H NMR: δ 2.34 (s, 3H), 3.83 (s, 3H), 6.59 (d, 1H, J=16.3 Hz), 6.90 (d,2H, J=8.7 Hz), 7.46 (d, 1H, J=16.3 Hz), 7.48 (d, 2H, J=8.7 Hz); ¹³C NMR:δ 27.5, 55.4, 114.4, 125.0, 127.1, 129.9, 143.1, 161.5, 198.1.

1-(4-Methoxymethyloxy-3-methoxyphenyl)-1-buten-3-one (35j).4-Methoxymethyloxy-3-methoxybenzaldehyde (1j, 2.30 g, 11.7 mmol) andacetone (19, 8.75 ml, 118.4 mmol) were combined in ethanol (20 ml) andstirred for 15 min at room temperature. A solution of sodium hydroxide(0.80 g, 20.0 mmol) and water (20 ml) was added and the mixture stirredfor 1 hr at room temperature. The resulting mixture was extracted intoethyl acetate, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford a solid. The crudesolid was recrystallized from hexane to give 2.70 g (97%) of a whitesolid: mp 73-75° C.; ¹H NMR: δ 2.34 (s, 3H), 3.48 (s, 3H), 3.89 (s, 3H),5.24 (s, 2H), 6.58 (d, 1H, J=16.1 Hz), 7.07 (m, 2H), 7.13 (d, 1H, J=8.7Hz), 7.43 (d, 1H, J=16.1 Hz); ¹³C NMR: δ 27.4, 55.9, 56.3, 95.2, 110.4,115.9, 122.5, 125.7, 128.7, 143.1, 148.7, 149.8, 198.0.

1-(2-Hydroxyphenyl)-1-buten-3-one (35q). 2-Hydroxybenzaldehyde (1q, 0.90ml, 8.4 mmol) and acetone (19, 1.24 ml, 16.8 mmol) were combined inethanol (7 ml) and stirred for 15 min at room temperature. A solution ofsodium hydroxide (0.5 g, 12.5 mmol) and water (2 ml) was added dropwiseand the mixture stirred for 48 hr at room temperature. The mixture wasneutralized with hydrochloric acid (1 N), extracted with ethyl acetate,washed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to afford a solid. The crude solid wasrecrystallized from tetrahydrofuran/hexane to give 0.36 g (26%) of ayellow solid: mp 136-137° C. [expected mp 139-140° C.]; ¹H NMR: δ 2.42(s, 3H), 6.92 (m, 2H), 7.03 (d, 1H, J=16.5 Hz), 7.24 (dt, 1H, J=7.0, 1.4Hz), 7.45 (d, 1H, J=7.7 Hz), 7.88 (d, 1H, J=16.3 Hz), 8.00 (s, 1H); ¹³CNMR: δ 26.8, 116.6, 120.5, 127.5, 129.5, 131.9, 141.0, 156.1, 156.1,201.3.

1-(4-Hydroxy-3-methoxyphenyl)-5-phenyl-1,4-pentadien-3-one (36a).1-(4-Methoxymethyloxy-3-methoxyphenyl)-1-buten-3-one (35j, 1.00 g, 4.2mmol) and benzaldehyde (1b, 0.46 ml, 4.5 mmol) were combined in ethanol(10 ml) and stirred for 15 min at room temperature. A solution of sodiumhydroxide (0.30 g, 7.5 mmol) and water (10 ml) was added and the mixturestirred for 18 hr at room temperature. The resulting mixture wasextracted into ethyl acetate, washed with saturated sodium chloride,dried over magnesium sulfate, filtered and evaporated to give 1.35 g(99%) of an oil which was used without purification. The oil (36j, 1.30g, 4.0 mmol) was stirred in methanol (50 ml) for 15 min at 60° C.Concentrated hydrochloric acid (3 drops) was added and the solutionstirred for 18 hr at 60° C. The methanol was evaporated and theresulting residue extracted into ethyl acetate, washed with saturatedsodium chloride, dried over magnesium sulfate, filtered and evaporatedto afford a semi-solid. The crude semi-solid was chromatographed onsilica gel with ethyl acetate/hexane to give 0.41 g (36%) of a yellowoil; ¹H NMR δ 3.92 (s, 3H), 6.08 (s, 1H), 6.91 (d, 1H, J=16.1 Hz), 6.93(d, 1H, J=8.2 Hz), 7.07 (d, 1H, J=15.9 Hz), 7.10 (s, 1H), 7.15 (d, 1H,J=8.0 Hz), 7.38 (m, 3H), 7.59 (m, 2H), 7.67 (d, 1H, J=15.9 Hz), 7.71 (d,1H, J=15.9 Hz); ¹³C NMR: δ 56.0, 109.8, 114.9, 123.4, 125.3, 127.3,128.3, 128.8, 130.3, 134.9, 142.8, 143.5, 146.8, 148.3, 188.7.

1-(4-Methoxyphenyl)-5-phenyl-1,4-pentadien-3-one (36e).1-(4-Methoxyphenyl)-1-buten-3-one (35e, 0.29 g, 1.6 mmol) was dissolvedin methanol (5 ml) and stirred for 5 min at room temperature. A solutionof sodium hydroxide (0.14 g, 3.5 mmol) and water (5 ml) was added andthe mixture stirred for 30 min at room temperature. Benzaldehyde (1b,0.17 ml, 1.7 mmol) was added dropwise and the mixture stirred for 18 hrat room temperature. The resulting precipitate was filtered andrecrystallized from ethanol to give 0.41 g (94%) of a yellow solid: mp85-89° C. [expected mp 118-119° C.]; ¹H NMR: δ 3.82 (s, 3H), 6.91 (d,2H, J=8.5 Hz), 6.94 (d, 1H, J=15.9 Hz), 7.06 (d, 1H, J=16.1 Hz), 7.38(m, 4H), 7.57 (m, 3H), 7.71 (dd, 2H, J=15.9, 2.0 Hz); ¹³C NMR: δ 55.4,114.4, 123.3, 125.5, 127.4, 128.2, 128.8, 130.0, 130.2, 134.8, 142.6,143.0, 161.5, 188.6.

2,6-Bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone (38a).2,6-Bis(4-methoxymethyloxy-3-methoxybenzylidene)cyclohexanone (38j, 0.49g, 1.1 mmol) was dissolved in methanol (100 ml) and stirred for 15 minat room temperature. Concentrated hydrochloric acid (3 drops) was addedand the mixture stirred for 3 hr at 60° C. The methanol was evaporatedand the resulting residue extracted into ethyl acetate, washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford a solid. The crude solid was recrystallized fromethanol to give 0.26 g (66%) of a yellow solid: mp 177-178° C. [expectedmp 179-181° C.]; ¹H NMR: δ 1.79 (m, 2H), 2.90 (t, 4H, J=5.4 Hz), 3.89(s, 6H), 5.88 (s, 2H), 6.91 (s, 2H), 6.96 (d, 2H, J=4.8 Hz), 7.06 (d,2H, J=8.0 Hz), 7.72 (s, 2H); ¹³C NMR: δ 23.1, 28.5, 56.0, 113.2, 114.4,124.4, 128.5, 134.2, 136.9, 146.2, 146.4, 172.8.

2,6-Bis(benzylidene)cyclohexanone (38b). Benzaldehyde (1b, 1.00 ml, 9.8mmol) and cyclohexanone (37, 0.51 ml, 4.9 mmol) were combined in ethanol(10 ml) and stirred for 15 min at room temperature. A solution of sodiumhydroxide (0.40 g, 10 mmol) and water (10 ml) was added and the mixturestirred for 18 hr at room temperature. The resulting precipitate wasfiltered and recrystallized from ethyl acetate to give 0.99 g (73%) ofyellow crystals: mp 118-119° C. [expected mp 117° C.]; ¹H NMR: δ 1.77(m, 2H), 2.92 (t, 4H, J=5.2 Hz), 7.39 (m, 10H), 7.80 (s, 2H); ¹³C NMR: δ23.1, 28.5, 128.3, 128.5, 130.2, 135.9, 136.1, 136.8, 190.1.

2,6-Bis(4-methoxymethyloxy-3-methoxybenzylidene)cyclohexanone (38j).4-Methoxymethyloxy-3-methoxybenzaldehyde (1j, 2.08, 10.1 mmol) andcyclohexanone (37, 0.55 ml, 5.3 mmol) were combined in ethanol (10 ml)and stirred for 15 min at room temperature. A solution of sodiumhydroxide (0.40 g, 10.0 mmol) and water (10 ml) was added and themixture stirred for 18 hr at room temperature. The resulting precipitatewas filtered and recrystallized from ethyl acetate to give 1.66 g (69%)of a yellow solid: mp 73-75° C.; ¹H NMR: δ 1.81 (m, 2H), 2.90 (t, 4H,J=5.2 Hz), 3.52 (s, 6H), 3.91 (s, 6H), 5.26 (s, 4H), 7.05 (m, 4H), 7.17(d, 2H, J=7.9 Hz), 7.74 (s, 2H); ¹³C NMR: δ 22.9, 28.4, 55.8, 56.1,95.1, 114.1, 115.6, 123.4, 130.2, 134.7, 136.4, 146.8, 149.1, 190.3.

1,5-Diphenylpentan-3-one (39b). 1,5-Diphenyl-1,4-pentadien-3-one (20b,1.00 g, 4.3 mmol) and palladium on activated carbon (0.25 g, 5%) werecombined in ethyl acetate (50 ml). The mixture was placed under ahydrogen atmosphere (60 psi) on a Parr apparatus for 2 hr at roomtemperature. The resulting mixture was filtered through celite and thesolvent evaporated to afford an oil. The crude oil was chromatographedon silica gel with ethyl acetate/hexane to give 0.82 g (80%) of a clearoil; ¹H NMR: δ 2.76 (t, 4H, J=7.6 Hz), 2.97 (t, 4H, J=7.4 Hz), 7.30 (m,10H); ¹³C NMR: δ 29.6, 44.2, 125.8, 128.0, 128.2, 140.7, 208.4.

1,5-Diphenylpentan-3-ol (40b). 1,5-Diphenyl-1,4-pentadien-3-one (20b,1.00 g, 4.3 mmol) and palladium on activated carbon (0.25 g, 5%) werecombined in ethyl acetate (50 ml). The mixture was placed under ahydrogen atmosphere (60 psi) on a Parr apparatus for 2 hr at roomtemperature. The resulting mixture was filtered through celite and thesolvent evaporated to afford an oil. The crude oil was chromatographedon silica gel with ethyl acetate/hexane to give 0.12 g (12%) of a whitesolid: mp 47-49° C. [expected mp 45-46° C.]; ¹H NMR: δ 1.86 (m, 4H),2.77 (m, 4H), 3.70 (m, 1H), 7.31 (m, 10H); ¹³C NMR: δ 32.1, 39.2, 70.8,125.6, 128.3, 142.0.

trans,trans-1,2,4,5-Diepoxy-1,5-diphenylpentan-3-one (42b) andcis,cis-1,2,4,5-diepoxy-1,5-diphenylpentan-3-one (43b). Potassiumfluoride dihydrate (9.40 g, 0.1 mol) and neutral aluminum oxide (10.0 g,98.1 mmol) were combined in water (100 ml) and stirred for 30 min atroom temperature. The water was evaporated and the resulting materialplaced in an oven for 5 days at 125° C. A suspension of potassiumfluoride-aluminum oxide (0.48 g, 3.0 mmol) in acetonitrile (6 ml) wasadded to a solution of 1,5-diphenyl-1,4-pentadien-3-one (20b, 0.47 g,2.0 mmol) in acetonitrile (1.0 ml) and the mixture stirred for 15 min atroom temperature. t-Butyl hydroperoxide (41, 1.7 ml, 17.7 mmol, 70%solution in water) was extracted with dichloroethane (6 ml), dried overmagnesium sulfate, filtered, added to the suspension and stirred for 30min at room temperature. The resulting mixture was filtered and thesolvent evaporated to afford a solid. The crude solid waschromatographed on silica gel with ethyl acetate/hexane to give amixture of isomers 42b and 43b. The crude solid was recrystallized twicefrom ethanol to give 0.21 g (39%) of 42b as white crystals: mp 117-119°C. [expected mp 118-118.5° C.]; ¹H NMR: δ 3.80 (d, 2H, J=1.4 Hz), 4.09(d, 2H, J=1.4 Hz), 7.30 (m, 10H); ¹³C NMR: δ 59.0, 60.9, 125.7, 128.7,129.2, 134.5, 199.0. The filtrate was evaporated to give 0.25 g (47%) of43b as a yellow oil; ¹H NMR: δ 3.72 (d, 2H, J=1.6 Hz), 4.18 (d, 2H,J=1.6 Hz), 7.33 (m, 10H); ¹³C NMR: δ 58.9, 60.3, 125.8, 128.7, 129.2,134.5, 199.0.

4-Methoxymethyloxy-3-methoxyacetophenone (44j).4-Hydroxy-3-methoxyacetophenone (44a, 2.5 g, 15 mmol) and potassiumcarbonate (15.0 g, 108.5 mmol) were combined in dimethyl formamide (50ml) and stirred for 15 min at room temperature. Chloromethyl methylether (18, 1.25 ml, 16.5 mmol) was added and stirring was continued for4 hr at room temperature. Potassium carbonate was filtered and thefiltrate extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated to 3.09g (98%) of an oil; ¹H NMR: δ 2.38 (s, 3H), 3.33 (s, 3H), 3.75 (s, 3H),5.12 (s, 2H), 6.99 (d, 1H, J=8.9 Hz), 7.35 (dd, 1H, J=6.6, 2.0 Hz), 7.82(s, 1H).

1,3-Bis(4-hydroxy-3-methoxyphenyl)-2-propen-1-one (45a).4-Methoxymethyloxy-3-methoxyacetophenone (44j, 2.14 g, 10.2 mmol) andbarium hydroxide octahydrate (3.25 g, 10.3 mmol) were combined inmethanol (50 ml) and stirred for 15 min at 50° C.4-Methoxymethyloxy-3-methoxybenzaldehyde (1j, 2.00 g, 10.2 mmol) wasadded and the mixture stirred for 18 hr at 50° C. The methanol wasevaporated and the resulting residue extracted into ethyl acetate,washed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to give 3.90 g (99%) of an oil which was usedwithout purification: ¹H NMR: δ 3.50 (s, 6H), 3.92 (s, 3H), 3.94 (s,3H), 5.25 (s, 2H), 5.30 (s, 2H), 7.18 (m, 4H), 7.39 (d, 1H, J=15.5 Hz),6.61 (m, 2H), 7.73 (d, 1H, J=15.5 Hz). The oil (45j, 1.10 g, 2.8 mmol)was stirred in methanol (50 ml) for 5 min at 60° C. Concentratedhydrochloric acid (3 drops) was added and the mixture stirred for 3 hrat 60° C. The methanol was evaporated and the resulting residue wasextracted into ethyl acetate, washed with saturated sodium chloride,dried over magnesium sulfate, filtered and evaporated to afford an oil.The crude oil was chromatographed on silica gel to give 0.47 (55%) of ayellow solid: mp 111-114° C. [expected mp 126-128° C.]; ¹H δ 3.94 (s,3H), 3.95 (s, 3H), 6.00 (s, 1H), 6.19 (s, 1H), 6.95 (m, 2H), 7.11 (d,1H, J=1.6 Hz), 7.20 (dd, 1H, J=8.3, 1.6 Hz), 7.38 (d, 1H, J=15.5 Hz),7.61 (m, 2H), 7.73 (d, 1H, J=15.7 Hz); ¹³C NMR: δ 56.0, 56.1, 110.0,110.5, 113.6, 114.8, 119.2, 123.0, 123.4, 127.6, 131.1, 144.2, 146.7,146.8, 148.0, 150.1, 188.4.

1,3-Diphenyl-propenone (45b). Acetophenone (44b, 1.20 ml, 10.3 mmol) andsodium hydroxide (0.40 g, 10.0 mmol) were combined in methanol (10 ml)and stirred for 30 min at room temperature. A solution of benzaldehyde(1b, 1.02 ml, 10.0 mmol) and methanol (10 ml) was added dropwise and themixture stirred for 21 hr at room temperature. Water (25 ml) was addedand the mixture neutralized with hydrochloric acid (1 N). The mixturewas extracted into ethyl acetate, washed with saturated sodium chloride,dried over magnesium sulfate, filtered and evaporated to afford asemi-solid. The crude semi-solid was chromatographed on silica gel withethyl acetate/hexane to give a solid. The solid was recrystallized fromhexane to give 1.11 g (53%) of a pale yellow solid: mp 52-54° C.[expected mp 55-58° C.]; ¹H NMR: δ 7.40 (m, 3H), 7.46 (t, 1H, J=1.6 Hz),7.63 (m, 5H), 7.81 (d, 1H, J=15.7 Hz), 8.02 (dd, 2H, J=8.0, 1.2 Hz); ¹³CNMR: δ 122.1, 128.3, 128.4, 128.5, 128.8, 130.4, 132.6, 134.8, 138.2,144.7, 190.3.

1-(4-Hydroxy-3-methoxyphenyl)-3-phenyl-2-propen-1-one (46a).4-Methoxymethyloxy-3-methoxyacetophenone (44j, 2.66 g, 12.7 mmol) andbarium hydroxide octahydrate (4.00 g, 12.7 mmol) were combined inmethanol (50 ml) and stirred for 5 min at 50° C. Benzaldehyde (1b, 1.30ml, 12.8 mmol) was added and the mixture stirred for 8 hr at 50° C. Themethanol was evaporated and the resulting residue was extracted intoethyl acetate, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to give 3.38 g (90%) of anoil which was used without purification; ¹H NMR: δ 3.48 (s, 3H), 3.92(s, 3H), 5.28 (s, 2H), 7.18 (d, 2H, J=8.9 Hz), 7.36 (m, 3H), 7.51 (d,1H, J=15.7 Hz), 7.60 (m, 3H), 7.77 (d, 1H, J=15.7 Hz). The oil (46j,3.35 g, 11.2 mmol) was stirred in methanol (75 ml) for 10 min at 50° C.Concentrated hydrochloric acid (3 drops) was added and the mixturestirred for 3 hr at 50° C. The methanol was evaporated and the resultingresidue was extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford an oil. The crude oil was distilled bulb to bulb to give 2.12(74%) of a yellow solid: mp 61-64° C. [expected mp 63-66° C.]; ¹H NMR δ3.95 (s, 3H), 6.29 (s, 1H), 6.98 (d, 1H, J=8.3 Hz), 7.38 (m, 2H), 7.53(d, 1H, J=15.5 Hz), 7.62 (m, 5H), 7.79 (d, 1H, J=15.5 Hz); ¹³C NMR: δ56.1, 110.5, 113.8, 121.6, 123.6, 128.3, 128.8, 130.2, 130.9, 135.0,143.8, 146.8, 150.4, 188.4.

1-(4-Carboxyphenyl)-3-phenyl-2-propen-1-one (46ad). 4-Acetylbenzonitrile(44al, 1.00 g, 6.9 mmol) and sulfuric acid (4 ml) were combined in water(4 ml) and the mixture stirred for 2.5 hr at reflux. The resultingmixture was extracted into ethyl acetate, washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated to give0.98 g (87%) of compound 44ad as a white solid: mp 204° C.; ¹H NMR:(DMSO) δ 2.61 (s, 3H), 8.04 (s, 4H), 13.23 (s, 1H). The solid (44ad,0.50 g, 3.0 mmol) and sodium hydroxide (0.29 g, 7.3 mmol) were combinedin water (4 ml) and ethanol (4 ml) and stirred for 30 min at roomtemperature. Benzaldehyde (1b, 0.31 ml, 3.1 mmol) was added and themixture stirred for 48 hr at room temperature. The resulting mixture wasacidified with hydrochloric acid (1 N), extracted into ethyl acetate,washed with saturated sodium chloride, dried over magnesium sulfate,filtered and evaporated to a afford a solid. The crude solid wasrecrystallized from ethyl acetate to give 0.54 g (70%) of a yellowsolid: mp 217-220° C. [expected mp 217-220° C.]; ¹H NMR: (DMSO) δ 7.45(m, 5H), 7.76 (d, 1H, J=16.1 Hz), 7.93 (d, 1H, J=15.5 Hz), 8.09 (d, 2H,J=7.9 Hz), 8.23 (d, 2H, J=7.6 Hz) 13.34 (s, 1H); ¹³C NMR: (DMSO) δ121.9, 128.5, 128.8, 128.9, 129.4, 130.6, 134.3, 134.4, 140.6, 144.6,166.4, 188.8.

1-(2,4-Dimethylphenyl)-3-phenyl-2-propen-1-one (46ak).2,4-Dimethylacetophenone (44ak, 1.48 g, 10.0 mmol) and sodium hydroxide(0.54 g, 13.5 mmol) were combined in methanol (30 ml) and stirred for 30min at room temperature. A solution of benzaldehyde (1b, 1.02 ml, 10.0mmol) and methanol (30 ml) was added dropwise and the mixture stirredfor 18 hr at room temperature. Water (25 ml) was added and the mixtureneutralized with hydrochloric acid (1 N). The mixture was extracted intoethyl acetate, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford an oil. The crudeoil was distilled bulb to bulb to give 1.98 g (84%) of a yellow oil:[expected mp 68° C.]; ¹H NMR δ 2.37 (s, 3H), 2.44 (s, 3H), 7.05 (m, 2H),7.16 (d, 1H, J=16.1 Hz), 7.38 (m, 3H), 7.49 (d, 1H, J=15.9 Hz), 7.54 (m,3H); ¹³C NMR: δ 20.4, 21.4, 126.0, 126.6, 128.2, 128.5, 128.8, 130.4,132.2, 134.7, 136.1, 137.4, 140.8, 145.0, 195.6.

1-(4-Cyanophenyl)-3-phenyl-2-propen-1-one (46al). 4-Acetylbenzonitrile(44al, 1.00 g, 6.9 mmol), sodium hydroxide (0.40 g, 10.0 mmol) and water(20 ml) were combined in ethanol (20 ml) and stirred for 15 min at roomtemperature. Benzaldehyde (1b, 0.70 ml, 6.9 mmol) was added and themixture stirred for 2 hr at room temperature. The resulting mixture wasfiltered and recrystallized from ethanol to give 1.46 g (91%) of ayellow solid: mp 120° C. [expected mp 119-120° C.]; ¹H NMR: δ 7.34 (m,3H), 7.62 (m, 2H), 7.80 (m, 4H), 8.06 (d, 2H, J=8.1 Hz); ¹³C NMR: δ115.9, 117.9, 121.1, 128.6, 128.8, 129.0, 131.0, 132.4, 134.3, 141.4,146.4, 188.9.

3-(4-Hydroxy-3-methoxyphenyl)-1-phenyl-2-propen-1-one (48a).4-Hydroxy-3-methoxybenzaldehyde (1a, 2.02 g, 13.3 mmol) andpyridiniump-toluenesulfonate (90 mg, 0.4 mmol) were combined indichloromethane (60 ml) and stirred for 5 min at room temperature. Asolution of 3,4-dihydropyran (47, 3.6 ml, 39.5 mmol) in dichloromethane(20 ml) was added dropwise and the mixture stirred for 5 hr at roomtemperature. The resulting mixture was washed with saturated sodiumchloride, dried over magnesium sulfate, filtered and evaporated toafford an oil. The crude oil was chromatographed on silica gel withethyl acetate/hexane to give 2.61 g (85%) of a clear oil. The oil (1am,1.01 g, 4.3 mmol) and barium hydroxide octahydrate (1.03 g, 3.3 mmol)were combined in methanol (26 ml) and stirred for 15 min at roomtemperature. Acetophenone (44b, 0.30 ml, 2.6 mmol) was added and themixture stirred for 16 hr at 50° C. The methanol was evaporated, waterwas added and the mixture acidified with hydrochloric acid (6 N). Theresulting mixture was extracted into ethyl acetate, washed withsaturated sodium chloride, dried over magnesium sulfate, filtered andevaporated to afford an oil. The crude oil was triturated with hexane togive a solid. The crude solid was recrystallized from ethylacetate/hexane to give 0.39 g (45%) of a yellow solid: mp 87-88° C. Thesolid (48am, 0.39 g, 1.2 mmol) andp-toluenesulfonic acid (0.10 g, 0.6mmol) were combined in methanol (50 ml) and stirred for 4 hr at roomtemperature. The methanol was evaporated and water was added. Themixture was neutralized with saturated sodium bicarbonate and extractedinto ethyl acetate. The ethyl acetate was washed with water, dried overmagnesium sulfate, filtered and evaporated to afford an oil. The crudeoil was chromatographed on silica gel with ethyl acetate/hexane toafford a solid. The crude solid was recrystallized from hexane to give0.18 g (62%) of a yellow solid: mp 81-84° C. [expected mp 85-90° C.]; ¹HNMR: δ 3.92 (s, 3H), 5.96 (s, 1H), 6.94 (d, 2H, J=8.1 Hz), 7.11 (s, 1H),7.21 (d, 1H, J=7.6 Hz), 7.35 (d, 1H, J=15.9 Hz), 7.51 (m, 2H), 7.73 (d,1H, J=15.5 Hz), 7.99 (d, 1H, J=7.0 Hz); ¹³C NMR: δ 56.1, 110.0, 114.8,119.8, 123.3, 127.4, 128.4, 128.5, 132.5, 138.5, 145.1, 146.7, 148.2,190.5.

3-(4-Carboxyphenyl)-1-phenyl-2-propen-1-one (48ad). Acetophenone (44b,0.50 ml, 4.3 mmol) and sodium hydroxide (0.50 g, 12.5 mmol) werecombined in ethanol (2 ml) and water (2 ml) and stirred for 30 min atroom temperature. 4-Formylbenzoic acid (1ad, 0.71 g, 4.7 mmol) was addedand the mixture stirred for 48 hr at room temperature. Water (25 ml) wasadded, the mixture acidified with hydrochloric acid (1 N) and theresulting precipitate was filtered and recrystallized from ethyl acetateto give 0.65 g (60%) of a white solid: mp 222-224° C. [expected mp227-229° C.]; ¹H NMR: (DMSO) δ 7.67 (m, 4H), 7.99 (m, 4H), 8.18 (m, 3H),13.14 (s, 1H); ¹³C NMR: (DMSO) δ 124.2, 128.5, 128.7, 128.8, 129.6,132.1, 133.2, 137.3, 138.7, 142.4, 166.7, 189.0.

1,3-Diphenylpropane-1,3-dione (50b). Methanol (0.26 ml, 6.4 mmol) andsodium (0.14 g, 6.1 mmol) were combined in xylene (60 ml) and stirredfor 20 min at room temperature. Methyl benzoate (49, 2.47 ml, 19.7 mmol)and acetophenone (0.58 ml, 5.0 mmol) were added and the mixture stirredfor 6 hr at 140° C. The mixture was cooled to room temperature andhydrochloric acid (10 ml, 6 N) was added and stirred for 15 min. Theresulting mixture was extracted into ethyl acetate, washed twice withwater, twice with saturated sodium bicarbonate and twice with water. Theethyl acetate was dried over magnesium sulfate, filtered and evaporatedto afford an oil. The crude oil was chromatographed on silica gel withethyl acetate/hexane to give a solid. The solid was recrystallized frommethanol to give 0.71 g (63%) of a pink-orange solid: mp 70-71° C.[expected mp 77-78° C.]; ¹H NMR: δ 6.85 (s, 1H), 7.51 (m, 6H), 7.98 (d,4H, J=6.8 Hz); ¹³C NMR: δ 93.1, 127.1, 128.6, 132.4, 135.5, 185.6.

2,6-Diphenyl-1-methyl-4-piperidone (52b).1,5-Diphenyl-1,4-pentadien-3-one (20b, 4.00 g, 17.1 mmol) was dissolvedin dimethyl formamide (60 ml). Methylamine (51, 6.0 ml, 70.0 mmol, 40%in water) was added and the mixture stirred for 96 hr at roomtemperature. The mixture was poured into water (250 ml) and stirred for1 hr at room temperature. The resulting mixture was extracted into ethylether, washed with saturated sodium chloride, dried over magnesiumsulfate, filtered and evaporated to afford a solid. The crude solid wasrecrystallized from ethanol to give 2.74 g (60%) of a white solid: mp147-149° C. [expected mp 148-150° C.]; ¹H NMR δ 1.82 (s, 3H), 2.50, (dd,2H, J=12.3, 2.5 Hz), 2.82 (t, 2H, J=13.3 Hz), 3.45 (dd, 2H, J=12.9, 2.4Hz), 7.34, (m, 10H); ¹³C NMR: δ 40.8, 50.8, 70.2, 127.0, 127.6, 128.8,143.1, 206.8.

2,6-Bis(2-methoxyphenyl)-1-methyl-4-piperidone (52c).1,5-Bis(2-methoxyphenyl)-1,4-pentadien-3-one (20c, 0.26 g, 0.9 mmol) wasdissolved in dimethyl formamide (5 ml). Methylamine (51, 0.40 ml, 4.6mmol, 40% in water) was added and the mixture stirred for 24 hr at roomtemperature. The mixture was poured into water (50 ml) and stirred for24 hr at room temperature. The resulting mixture was extracted intoethyl acetate, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford a solid. The solidwas recrystallized twice from ethanol to give 0.16 g (55%) of a whitesolid: mp 146-148° C.; ¹H NMR δ 1.89 (s, 3H), 2.50 (d, 2H, J=13.7 Hz),2.65 (t, 2H, J=11.9 Hz), 3.82 (s, 6H), 4.11 (d, 2H, J=11.5 Hz), 6.87 (d,2H, J=8.3 Hz), 7.03 (t, 2H, J=7.2 Hz), 7.23 (t, 2H, J=5.8 Hz), 7.72 (d,2H, J=7.6 Hz); ¹³C NMR: δ 40.3, 49.2, 55.4, 61.2, 110.7, 121.0, 127.6,127.8, 131.5, 156.3, 208.1; Exact mass calcd for C₂₀H₂₃NO₃: 325.1678,observed (M+H) 326.1754.

2,6-Bis(4-methoxyphenyl)-1-methyl-4-piperidone (52e).1,5-Bis(4-methoxyphenyl)-1,4-pentadien-3-one (20e, 0.40 g, 1.4 mmol) wasdissolved in dimethyl formamide (10 ml). Methylamine (51, 0.75 ml, 8.7mmol, 40% in water) was added and the mixture stirred for 24 hr at roomtemperature. The mixture was poured into water (50 ml) and stirred for 2hr at room temperature. The resulting mixture was extracted into ethylacetate, washed with saturated sodium chloride, dried over magnesiumsulfate, filtered and evaporated to afford a solid. The crude solid waschromatographed on silica gel with ethyl acetate/hexane to afford asolid that was recrystallized from ethanol to give 0.30 g (68%) of awhite solid: mp 141-143° C. [expected mp 129-130° C.]; ¹H NMR δ 1.77,(s, 3H), 2.45 (d, 2H, J=14.5 Hz), 2.78 (t, 2H, J=12.9 Hz), 3.33 (d, 2H,J=11.9 Hz), 3.79 (s, 6H), 6.88 (d, 4H, J=8.5 Hz), 7.32 (d, 4H, J=8.5Hz); ¹³C NMR: δ 40.6, 50.9, 55.3, 69.5, 114.1, 128.0, 135.3, 158.9,207.1.

2,6-Bis(4-methylphenyl)-1-methyl-4-piperidone (52aa).1,5-Bis(4-methylphenyl)-1,4-pentadien-3-one (20aa, 0.32 g, 1.2 mmol) wasdissolved in dimethyl formamide (9 ml). Methylamine (51, 0.5 ml, 5.8mmol, 40% in water) was added and the mixture stirred for 72 hr at roomtemperature. The mixture was poured into water (50 ml) and stirred for 2hr at room temperature. The resulting mixture was extracted into ethylacetate, washed with saturated sodium chloride, dried over magnesiumsulfate, filtered and evaporated to afford a solid. The crude solid waschromatographed on silica gel with ethyl acetate/hexane to afford asolid that was recrystallized from ethanol to give 0.26 g (75%) of awhite solid: mp 120-121° C. [expected mp 105-107° C.]; ¹H NMR δ 1.79 (s,3H), 2.36 (s, 6H), 3.10 (d, 2H, J=14.9 Hz), 2.79 (t, 2H, J=13.1 Hz),3.35 (dd, 2H, J=11.9, 2.4 Hz), 7.15 (d, 4H, J=8.0 Hz), 7.21 (d, 4H,J=7.9 Hz); ¹³C NMR: δ 21.2, 40.7, 50.9, 70.0, 126.9, 129.4 137.2, 140.2,207.1.

2,6-Bis(2-methylphenyl)-1-methyl-4-piperidone (52ac).1,5-Bis(2-methylphenyl)-1,4-pentadien-3-one (20ac, 0.50 g, 1.9 mmol) wasdissolved in dimethyl formamide (10 ml). Methylamine (51, 1.0 ml, 11.6mmol, 40% in water) was added and the mixture stirred for 24 hr at roomtemperature. The mixture was poured into water (50 ml) and stirred for 2hr at room temperature. The resulting mixture was extracted into ethylacetate, washed with saturated sodium chloride, dried over magnesiumsulfate, filtered and evaporated to afford a solid. The crude solid waschromatographed on silica gel with ethyl acetate/hexane to afford asolid that was recrystallized from ethanol to give 0.29 g (52%) of awhite solid: mp 155-157° C.; ¹H NMR δ 1.82, (s, 3H), 2.40, (s, 6H), 2.44(d, 2H, J=11.9 Hz), 2.77 (t, 2H, J=13.3 Hz), 3.74 (d, 2H, J=11.9 Hz),7.16 (m, 6H), 7.67, (d, 2H, J=7.0 Hz); ¹³C NMR: δ 19.5, 39.9, 49.3,65.8, 126.7, 126.9, 130.6, 134.8, 140.9, 207.2; Exact mass calcd forC₂₀H₂₃NO: 293.1779, observed (M+H) 294.1856.

2,6-Bis(2-naphthyl)-1-methyl-4-piperidone (53).1,5-Bis(2-naphthyl)-1,4-pentadien-3-one (25, 0.82 g, 2.5 mmol) wasdissolved in dimethyl formamide (15 ml). Methylamine (51, 1.30 ml, 15.1mmol, 40% in water) was added and the mixture stirred for 72 hr at roomtemperature. The mixture was poured into water (100 ml) and stirred for24 hr at room temperature. The resulting mixture was extracted intoethyl acetate, washed with saturated sodium chloride, dried overmagnesium sulfate, filtered and evaporated to afford a solid. The crudesolid was chromatographed on silica gel with ethyl acetate/hexane toafford a solid that was recrystallized twice from ethanol to give 0.20 g(22%) of a white solid: mp 209-212° C.; ¹H NMR δ 1.89 (s, 3H), 2.59 (d,2H, J=13.9 Hz), 2.97 (t, 2H, J=11.3 Hz), 3.66 (d, 2H, J=11.7 Hz), 7.49(m, 4H), 7.81 (m, 10H); ¹³C NMR: δ 41.1, 50.7, 70.3, 124.6, 125.9,126.0, 126.2, 127.6, 127.7, 128.9, 133.0, 133.4, 140.4, 206.6; Exactmass calcd for C₂₆H₂₃NO: 365.1780, observed (M+H) 366.1852.

Example 2 Antioxidant Activity of Curcumin Derivatives

It has been suggested that the antioxidant activity of curcumin dependson the phenolic groups (Barclay et al., Organic Lett. 2(18), 2841-2843(2000); Priyadarsini et al., Free Radical Biol. Med. 35(5), 475-484(2003)). However, other studies support the conclusion that the centralmethylene hydrogens of curcumin are important for antioxidant activity(Jovanovic et al., J. Am. Chem. Soc. 123(13), 3064-3068 (2001)). Morerecently it has been demonstrated that both the central methylenehydrogens and the phenolic hydrogens may be involved in the mechanism offormation of the phenoxy radical, depending upon reaction conditions(Litwinienko et al., J. Org. Chem. 69(18), 5888-5896 (2004)). Thelibrary consisting of three series of analogs examined the role of theenone functionality in aryl systems where the spacer is 7-carbons (as incurcumin), 5-carbons or 3-carbons in length. In addition, the importanceof aryl ring substituents including phenolic groups was assessed as wellas the importance of the central methylene hydrogens of curcumin. Theantioxidant activities of the curcumin analogs were determined in twostandard assays. There are multiple standardized methods to determineantioxidant activities, and it is recommended that at least twodifferent procedures be used (Barclay et al., Organic Lett. 2(18),2841-2843 (2000)). The first assay was the Total Radical-trappingAnti-oxidant Parameter assay (TRAP assay) and the second assay was theFerric Reducing/Anti-oxidant Power assay (FRAP assay).

TRAP Assay

The first procedure called for antioxidant activity to be measured asthe ability of the analogs to react with the pre-formed radicalmonocation of 2,2′-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid(ABTS⁺). This assay is also known as the Total radical-trappinganti-oxidant parameter assay (TRAP assay). For the TRAP assay (Re etal., Free Rad. Biol. Med. 26, 1231-1237 (1999)),2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS, 1.8 mM) wasreacted with potassium persulfate (0.63 mM) in double distilled water,at room temperature in the dark, overnight, to generate the dark bluecolored ABTS⁺ radical cation, which has a maximum absorption at 734 nm.Just before the experiment, ABTS⁺ was diluted with absolute ethanol toan absorbance of approximately 0.7 at 734 nm. ABTS⁺ (1 ml) was added tocurcumin or its analogs (10 μM in ethanol) and mixed by vortexing. Theturquoise colored reaction was allowed to stabilize for 5 min and theabsorbance monitored on a Perkin Elmer UV/Vis Lambda 2S. The activitiesof curcumin and its analogs were determined by their abilities to quenchthe color of the radical cation. The synthetic analog of α-tocopherol(vitamin E), Trolox, was used as a reference standard (10 μM inethanol).

The first assay, the TRAP assay, determines the analogs abilities toreduce a radical cation generated from2,2′-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS). Thefollowing FIGS. (2A-2C) show the analogs active in the TRAP assay. Theactive analogs in FIGS. 2A-2C are arranged from highly active on theleft to slightly active on the right.

Active analogs retaining a 7-carbon spacer as in curcumin are shown inFIG. 3A. Four analogs, 13a, 14a, 15a and 3h, in this series were foundto be more active than curcumin (3a). Of the ten active analogs in thisseries, eight retain phenolic groups as in curcumin. The three bestanalogs, 13a, 14a and 15a, not only contained phenolic groups but alsocontained a saturated spacer between the aryl rings. It is also evidentthat a central methylene substituent is favorable in analogs displayingantioxidant activity as six of the ten analogs in FIG. 3A contain acentral methylene substituent.

Active analogs in series 2, which have a 5-carbon spacer, are shown inFIG. 3B. Two analogs, 20af and 20q, in this series were found to be moreactive than curcumin. Of the six active analogs in this series, fivecontain phenolic groups. The best analog, 20af, is a tetraphenol.

Active analogs in series 3, which have a 3-carbon spacer, are shown inFIG. 3C. Only one analog, 45a, in this series was found to be moreactive than curcumin. All five of the active analogs in this seriescontain phenolic groups.

FRAP Assay

Anti-oxidant activity was also measured in the Ferricreducing/anti-oxidant power assay (FRAP assay) in which the analogs arereacted with a ferric tripyridyltriazine complex. For the FRAP assay(Benzie et al., Meth. Enzymol. 1999, 299, 15-27), the ferric complex wasprepared at room temperature by reaction of ferric chloride (16.7 mM)and 2,4,6-tripyridyl-s-triazine (8.33 mM) in acetate buffer (0.25 M) topH 3.6. The FRAP reagent was used immediately after preparation. Theferric complex (1 ml) was added to curcumin or its analogs (10 μM inethanol). The reaction was mixed by vortexing, allowed to stabilize for5 min and the absorbance recorded on a Perkin Elmer UV/Vis Lambda 2S.The activities of curcumin and its analogs were determined by theirabilities to reduce the ferric complex to a ferrous complex, monitoredby the formation of the purple colored ferrous complex at 593 nm. Thesynthetic analog of α-tocopherol (vitamin E), Trolox, was used as areference standard (10 μM in ethanol).

The second assay used, the FRAP assay, determines the analogs abilitiesto reduce a Fe(III) tripyridyltriazine complex to a Fe(II)tripyridyltriazine complex. The following FIGS. (3A-3C) show analogsactive in the FRAP assay. The active analogs in FIGS. 3A-3C are arrangedfrom highly active on the left to slightly active on the right.

Active analogs retaining a 7-carbon spacer as in curcumin are shown inFIG. 4A. Curcumin (3a) displayed the most antioxidant activity in thisseries. Analog 13a, the reduced form of curcumin, also displayed potentantioxidant activity. Six of the eight active analogs in this seriescontain phenolic groups as in curcumin and four analogs containsubstituents on the central methylene carbon.

Active analogs in series 2 are shown in FIG. 4B. Only one analog, 20af,in this series was found to be more active than curcumin. The two bestanalogs, 20af and 38a, in this series contain phenolic groups. However,contrary to any of the previous antioxidant results, only three of thetwelve most active analogs in this series contain phenolic groups. Thereis currently no explanation as to why nine of the top twelve analogs inthis series contain no phenolic groups and further investigation isnecessary.

Active analogs in series 3 are shown in FIG. 4C. No analog in thisseries was more active than curcumin. Three of the five active analogs,45a, 35a and 48a, in this series contain phenolic groups and a fourth,46ad, contains an acidic carboxylic acid proton.

Most analogs that display antioxidant activity retain phenolic groups.Eighteen of the twenty one active analogs in the TRAP assay and twelveof the seventeen active analogs (minus the eight least active in the5-carbon series) contain phenolic groups. This indicates that a phenolicsubstituent is desirable for antioxidant activity but not essential.Analogs in all three series were found to contain antioxidant activitywith seven analogs in the 7-carbon series, three in the 5-carbon seriesand four in the 3-carbon series displaying activity in both the TRAP andFRAP assays.

Example 3 Inhibition of NF-κB Activity by Curcumin Derivatives

Curcumin and its analogs were screened for activity against NF-κB by acellular assay using the NF-κB stable cell line (293T/NF-κB-luc). Thecell line is derived from human 293T embryonic kidney cells expressingthe large T antigen containing a chromosomal integration of a luciferasereporter construct regulated by 6 copies of the NF-κB response element(Panomics, Inc.). This stable clonal cell line is obtained byco-transfection of pNF-κB-luc and pTK-hyg containing plasmids followedby the addition of hygromycin (200 μg/ml) to maintain cell selection.

The cell line was grown in a humidified atmosphere at 37° C. in 5%CO₂/95% air and maintained in Dulbecco's Modified Eagle's Medium(DMEM-high glucose containing 4 mM glutamine) containing fetal bovineserum (FBS, 10%), sodium pyruvate (1 mM), penicillin (100 units/ml),streptomycin (100 μg/ml) and hygromycin (100 μg/ml) to maintain cellselection (Gibco/Invitrogen).

The 293T/NF-κB-luc cells were re-plated 24 hr prior to treatment, into24-well cell culture plates in media without hygromycin, to prevent itfrom interfering with the assay. The cells were then allowed to grow andattach, to the wells, for 24 hr in a humidified atmosphere at 37° C. in5% CO₂/95% air. After 24 hr, the cells had reached approximately 70%confluency and were given fresh media (1 ml) 1 hr prior to treatmentwith curcumin and its analogs. The cells were then re-given media (1 ml)with or without recombinant tumor necrosis factor alpha (TNFα, 20 ng/mlin phosphate buffered saline (PBS) at pH 7.4 containing 0.1% human serumalbumin, R&D Biosciences/Clontech) followed by immediate treatment withcurcumin or its analog (10 μM in DMSO). The cells then were placed againin a humidified atmosphere at 37° C. in 5% CO₂/95% air for 7 hr. Platewells were gently washed with PBS, pH 7.4, and lysed with passive lysisbuffer (1x, 100 μl, Promega). The subsequent chemiluminescent lysateswere analyzed with the Luciferase Assay System (Promega) utilizing aTD-20/20 luminometer. The relative light units (photons) were determinedby the addition of firefly luciferase substrate (75 μl) to cell lysate(10 μl). The light units were then normalized to the amount of proteinin the well (mg/ml) with BCA Protein Assay Kit (Pierce) and standardizedto percent of control (TNFα).

To determine cell viability, cells were treated as above but with 15 μManalog. After gently washing to remove any dead cells, they were givenmedia (100 μl) and CellTiter 96® AQueous One Solution reagent (20 μl)for 1 hour and read at 490 nm with a Spectromax plate reader.

Curcumin is a known inhibitor of the NF-κB activation cascade.Therefore, modification of the structure of curcumin could lead toenhanced activity. The library consisting of three series of curcuminanalogs were used to examine the role of the enone functionality in arylsystems where the spacer is 7-carbons (as in curcumin), 5-carbons or3-carbons in length. In addition, the importance of aryl ringsubstituents was assessed. The NF-κB activities of curcumin and analogswere determined by a cellular firefly luciferase assay. This assayutilized a commercially available cell line (Panomics 293T-luc cellularassay) developed for screening inhibitors of NF-κB. This cell line isstably transfected with a luciferase reporter controlled by an NF-κBdependent promoter. The cell is stimulated with tumor necrosis factoralpha (TNFα) which activates NF-κB. NF-κB then binds to one of sixpromoter regions on the cell's DNA leading to the production of aluciferase enzyme. Luciferin is added to the cell lysates and theluciferase enzyme catalyzes a cleavage of luciferin leading to theemission of light.

The following FIGS. (4A-4C) show analogs active in the NF-κB cellularassay. The active analogs in FIGS. 4A-4C are arranged from highly activeon the left to slightly active on the right.

Active analogs in series 1, which contain a 7-carbon spacer, are shownin FIG. 5A. Three analogs, 9a, 6a and 14a in this series were moreactive than curcumin (3a). These three analogs all contain the same arylsubstituents as in curcumin. In addition, five of the six best analogsin this series contain a substituent on the central methylene carbon,indicating this position may be important to enhance activity. Threeanalogs also contain a saturated 7-carbon spacer indicating thatsaturation may be important in this series. Four of the six activeanalogs in this series have antioxidant activity. It is important tonote that two analogs were active against NF-κB activation independentof antioxidant activity.

Active analogs in series 2, which contain a 5-carbon spacer, are shownin FIG. 5B. Ten analogs, 29, 38a, 20v, 31, 20a, 20ag, 20q, 20w, 20m and20o in this series were more active than curcumin. Eight of the tenactive analogs in this series contain aryl substituents. Six analogscontain substituents meta to the spacer on the aryl group, indicatingthis position may be important for NF-κB activity. Analogs 29 and 31contain pyridine rings with no substituents on the ring. These twoactive analogs indicate that if the analogs in this series have aspecific target, the target may contain a hydrogen bond donor in thearea of binding. Only one of the ten active analogs in this seriesdisplays antioxidant activity. Therefore, it can then be concluded thatthese analogs are targeting a specific protein.

Active analogs in series 3, which contain a 3-carbon spacer, are shownin FIG. 5C. Three analogs, 45a, 52b and 35a, in this series were moreactive than curcumin. Three of the seven active analogs in this seriesretain the same aryl substituents as in curcumin. Analog 52b contains apiperidone ring on the spacer, indicating this type of spacer may beimportant for activity. Two of the seven active analogs were active asantioxidants. It is important to note that five analogs were activeagainst NF-κB independent of antioxidant activity.

The IC₅₀ values for the active analogs against NF-κB were also measured.An IC₅₀ value is the concentration of the analog necessary to give 50%inhibition of NF-κB activation. The IC₅₀ plot for curcumin is shown inFIG. 6A. IC₅₀ plots for additional active analogs are shown in FIGS.6B-6L. Table 5 shows IC₅₀ values for eight of the active analogs fromthe screening assay. Table 5 also shows if each analog was active as anantioxidant (+) in both the TRAP and FRAP assays. Of the IC₅₀ valuesobtained, curcumin (8.2 μM) is the least potent analog against NF-κB.Analogs 29 and 31 which contain pyridine rings are the most activeanalogs against NF-κB with IC₅₀ values of 3.5 and 3.4 μM. As observed inTable 5, five analogs are active against NF-κB independent ofantioxidant activity. This indicates that the analogs are targetingspecific proteins in the cell.

TABLE 5 IC₅₀ Values and Antioxidant Results for Active Analogs AgainstNF-κB. Analog IC₅₀ Structure Number (μM) TRAP FRAP

31 3.4 − −

29 3.5 − −

38a 4.2 + +

20q 4.2 + −

20ag 5.4 − +

20m 6.4 − −

6a 6.8 + +

9a 7.6 + +

Example 4 Molecular Modeling of Curcumin Derivatives Binding to NF-κB

Molecular modeling studies can be performed to obtain useful informationfor the design of potent analogs. Modeling allows the visualization ofligand-protein interactions which can identify a potential inhibitorbinding site in a protein. In most cases the substrate binding site isknown from crystal structures that contain the native substrate or asubstrate analog. Binding sites are also identified through crystalstructure data involving bound inhibitors. Removal of the knowninhibitor and addition of a potential inhibitor can give usefulinformation concerning inhibitor protein interactions as well asestimated inhibition constants. Estimated inhibition constants (K_(est))can be obtained from the docking studies with the modeling program.These constants can be compared to experimentally obtained inhibitionconstants (K_(exp)). If a correlation between K_(est) and K_(exp) isfound then a new potential inhibitor can be docked to obtain K_(est) todetermine if synthesis of the analog is warranted.

The dockings were performed using the docking program Autodock 3.0(Morris et al., J. Comp. Chem. 19(14), 1639-1662 (1998); Morris et al.,J. Comput.-Aided Mol. Des. 10(4), 293-304 (1996)) on a cluster ofSilicon Graphics workstations consisting of Octanes and O2s. Theanalogs, prepared using Sybyl 7.0 (Tripos Inc.), were drawn, assignedpartial charges using the included Gasteiger-Hückel method and energyminimized using the Broyden, Fletcher, Goldfarb and Shanno (BFGS)optimization method. Minimizations were run for 10,000 iterations andall rotateable bonds were defined before docking. The proteins wereprepared before docking in Sybyl by removing non-native substrates andwater molecules. Polar hydrogens and Kollman Uni charges were added tothe proteins as well. The molecules were docked in an area definedaround the protein as a cube of either 60×60×60 Å or 120×120×120 Å.

Since the protein target of the analogs is unknown, molecular modelingwas employed to examine a possible correlation between K_(est) andK_(exp). On proteins such as HSP90 (protein data bank code 1YER and1YES) and glutathione S-transferase (19GS) the location of the analogbinding site is known. Docking studies were performed using Autodock 3.0and the resulting K_(est) was compared to K_(exp).

On proteins such as NF-κB (1IKN and 1SCV) and AP-1 (1FOS), the locationof analog binding site is not known. Therefore, it was necessary toidentify any and all potential binding areas and model the analogs tothese areas. Fortunately, a program has been developed that can identifybinding areas (Brown et al., J. Chem. Inf. Comp. Sci. 44(4), 1412-1422(2004)). The program called the Macromolecule Encapsulating Surface(MES) program generates a flexible surface over the entire protein anddetermines how much unoccupied volume there is between the generatedsurface and the surface of the protein. If there is a large space, thatarea is a potential binding site and it is possible for a potentialinhibitor to fill this space. On the other hand, if there is no spacethe program overlooks that area and dismisses it from futureconsideration. Once all of the potential binding areas are identified,the program will dock inhibitors in each of these locations anddetermine the K_(est) for each analog.

Binding to NF-κB

When performing docking studies of the potential analogs against NF-κB,two crystal structures, 1IKN and 1SVC, were selected from the twelveavailable in the protein databank. These two crystal structures wereselected because one (1IKN) contained both the p50 and p65 subunits ofNF-κB. The other crystal structure was selected because it contained thep50 subunit of NF-κB bound to a short segment of DNA.

Binding to the 1IKN Form of NF-κB

1IKN (Huxford et al., Cell 1998, 95, 759-770) was selected because itcontains both the p50 and p65 subunits of NF-κB, the most commonheterodimer. The p50 subunit was not the complete subunit. A secondreason 1IKN was selected was because it contained I-κB, the naturalinhibitor of NF-κB. Since I-κB is phosphorylated at serine residues 32and 36, in the activation cascade of NF-κB, it was hoped that thecrystal structure would contain these residues to see if the potentialanalogs blocked them from being phosphorylated. Unfortunately, thecrystal structure of I-κB did not contain these residues and thusdocking studies could not be performed directly on the I-κB subunit.FIG. 7 shows the p50 and p65 NF-κB heterodimer complexed to I-κB. InFIG. 7, the blue protein is the p50 subunit, the red protein is the p65subunit and the yellow protein is I-κB.

When I-κB is removed as shown in FIG. 8, a new face of the heterodimeris revealed. It is believed that DNA binds to this new face of theprotein after NF-κB translocates to the nucleus. If the potentialanalogs inhibit NF-κB from binding to DNA and thus stoppingtranscription from occurring, then the potential analogs should bind tothis face of the molecule. However, only analogs 9a, 9b, 12b, 15a, 15b,17b, 20l and 52l of the analog library bind to the newly exposed face asshown in FIG. 9. Analogs 12b, 15a and 17b have a good K_(est) values andrank in the top nine analogs. Analog 12b binds to NF-κB on this newlyexposed face of the molecule and the K_(est) value is good at 2.00E-10M. These results indicate the analogs should be blocking the NF-κB-DNAinteraction. However, there is no correlation between the analogs thatbind on this face of the molecule and the experimental results of theseanalogs. Based on the docking studies, it does not appear that theanalogs block a NF-κB-DNA interaction, but it is possible that theyinhibit NF-κB in another manner.

Curcumin (3a), shown in FIG. 10, and the other potential inhibitors bindon the opposite side of the molecule as shown in FIG. 11. Many of theanalogs that bind on the opposite side also have good K_(est) valueswith analogs 3i, 20ag, 23 and 53 being the best. Table 6 shows eachanalog with its K_(est) value in molar units. Again, there is nocorrelation between K_(est) and K_(exp). This indicates that thepotential inhibitors probably do not bind to the NF-κB heterodimer.

TABLE 6 K_(est) Values of Curcumin Analogs Against NF-κB Without MES.12b 2.00E−10 11b 3.31E−09 20q 9.59E−09 46a 3.07E−08 20ag 3.07E−10 20v3.38E−09 20ah 9.80E−09 20r 3.16E−08 17b 3.27E−10 52e 3.52E−09 20z1.11E−08 31 3.66E−08 53 3.72E−10  9b 4.09E−09 45a 1.20E−08 29 4.87E−08 3i 6.31E−10 36a 4.55E−09 40af 1.29E−08 46al 5.03E−08 23 7.36E−10  6a4.62E−09 20x 1.35E−08 46ak 5.16E−08 20n 7.86E−10 20w 5.08E−09  3c1.39E−08 39b 5.32E−08 25 1.16E−09 20o 5.32E−09 20ab 1.63E−08 20b5.82E−08 15a 1.20E−09 20a 5.42E−09 13b 1.68E−08 20s 6.08E−08  3d1.34E−09 20p 5.42E−09 20ae 1.75E−08 20f 6.66E−08 14a 1.34E−09 16b5.71E−09 52b 1.76E−08 42b 6.78E−08  9a 1.51E−09 20k 6.38E−09 38b1.87E−08 20l 7.00E−08  3b 1.94E−09 52aa 6.52E−09 48a 1.92E−08 50b7.12E−08  3g 2.32E−09 52ac 7.38E−09 20t 2.35E−08 34 9.37E−08 38a2.36E−09  3f 7.60E−09 36e 2.39E−08 40b 1.23E−07 14b 2.38E−09 20d7.74E−09 20g 2.45E−08 43b 1.41E−07 20m 2.49E−09 20c 7.85E−09  3e2.54E−08 52l 1.76E−07 15b 2.89E−09  3h 8.02E−09 20e 2.60E−08 45b2.19E−07  3a 2.94E−09 46ad 8.96E−09 48ad 2.71E−08 35a 5.30E−07  6b3.06E−09 20y 9.03E−09 20aa 2.85E−08 35e 1.66E−06 13a 3.17E−09 20u9.53E−09 20ac 2.90E−08 35q 3.53E−06 20i 3.29E−09

To verify these findings, the MES program was utilized on the NF-κBheterodimer to identify any potential binding areas for the analogs. Theresults of this docking study are different than the docking resultsobtained without the use of the MES program. With the MES program, allthe potential inhibitors bind on the new face of the NF-κB heterodimeras shown in FIG. 12. The visual results of this docking study indicatethat the analogs should be good inhibitors of NF-κB and in particular ofa NF-κB-DNA interaction. Most of the potential inhibitors bind to NF-κBwith good Kes,t values with analogs 9a, 12b, 13a, 15a and 20ag showingthe best activity as shown in Table 7. Curcumin (3a), as shown in FIG.13, binds to the heterodimer towards the bottom portion of the p50subunit and has a K_(est) value of 9.64E-8 M. However, the K_(est)values of curcumin and its analogs do not correlate to K_(exp) values,they probably do not bind to NF-κB.

TABLE 7 K_(est) Values for NF-κB (1IKN) with MES. 20ag 7.26E−09 40af8.72E−08  6b 2.19E−07 48ad 7.60E−07  9a 7.39E−09 14b 8.86E−08 20y2.22E−07 20t 8.03E−07 15a 1.26E−08 20k 9.43E−08 13c 2.41E−07 20l8.15E−07 12b 1.54E−08  3a 9.64E−08 52e 2.47E−07 50b 8.72E−07 13a1.57E−08 15b 9.69E−08 20p 2.56E−07 46ak 9.22E−07 17b 1.65E−08 20u9.88E−08 52l 2.57E−07 42b 9.44E−07  9b 1.93E−08 36a 1.06E−07 20aa2.71E−07 40b 9.64E−07 25 2.51E−08 20w 1.29E−07  3e 2.74E−07 29 1.03E−06 3d 2.59E−08  3h 1.40E−07 20x 2.81E−07 52b 1.08E−06 20ae 2.65E−08  3g1.47E−07 20ab 2.82E−07 46al 1.14E−06 20i 2.69E−08 46ad 1.56E−07 20z3.78E−07 46a 1.24E−06 38a 3.27E−08 20o 1.63E−07 52aa 3.84E−07 20b1.25E−06 20a 4.38E−08 20f 1.74E−07  3b 4.03E−07 31 1.33E−06 20ah5.27E−08  3i 1.82E−07 20q 4.16E−07 39b 1.42E−06 20m 5.61E−08 16b1.82E−07 20c 4.46E−07 43b 2.01E−06 53 6.06E−08 36e 1.86E−07  3f 4.47E−0738b 2.41E−06 14a 6.10E−08 20d 1.86E−07 52ac 4.61E−07 45b 4.15E−06 20v6.66E−08 20g 1.86E−07 20ac 4.92E−07 34 4.22E−06 23 7.21E−08 20e 1.87E−0720s 5.49E−07 35a 4.46E−06 11b 7.35E−08 13b 2.03E−07 20r 6.43E−07 35e4.73E−06 20n 8.21E−08 45a 2.05E−07 48a 6.44E−07 35q 4.81E−06  6a8.63E−08Binding to the 1SVC Form of NF-κB

1SVC (Mueller et al., Nature, 373, 311-317 (1995)) was selected becauseit contains the p50 subunit of NF-κB bound to a small portion of DNA.This crystal structure was important because it contained almost all ofthe p50 subunit and because it had the exact site of DNA binding. Thiswould provide additional information concerning the blocking ofNF-κB-DNA binding interactions of the analogs. FIG. 14 shows the p50subunit of NF-κB bound to a small portion of DNA. In FIG. 14, the blueprotein is the p50 subunit and the yellow segment is the DNA. When theDNA is removed as shown in FIG. 15, a new area is exposed. It is in thislocation that the analogs will bind if they are preventing a NF-κB-DNAbinding interaction.

When docking studies were performed, most of the potential inhibitorsbind in the general area the DNA once occupied as shown in FIG. 16. Itis apparent that in the location of binding, there is a “small hole” towhich all of the analogs on this portion of the molecule bind. FIG. 17shows curcumin (3a) bound in the “small hole”.

Many of these analogs bind with good K_(est) values with analog 9bdisplaying the best inhibitory activity at 3.79E-10 M as shown in Table8. Based on these K_(est) values, several analogs should inhibit theblocking of NF-κB-DNA binding interactions. However, there is nocorrelation to the K_(exp) results indicating that these analogsprobably do not inhibit this type of an interaction. It is possible thatthere could be another mode of action that potential inhibitors could bedisplaying since seven analogs bind on the opposite side of the proteinas shown in FIG. 18. These seven analogs, 12b, 15a, 15b, 52e, 52l, 52aaand 52ac have rather poor K_(est) values with the exception of analog12b which was ranked as the third best potential inhibitor. Since theseanalogs have poor K_(est) values and there is no correlation to anyK_(exp) results, they are likely not inhibitors of the NF-κB protein.

TABLE 8 K_(est) Values for NF-κB (1SVC).  9b 3.79E−10 20ag 1.32E−08 20w4.68E−08 20b 1.57E−07  3g 1.59E−09 20u 1.33E−08 20ac 4.70E−08 291.65E−07 12b 3.30E−09 20o 1.34E−08 53 4.70E−08 48ad 1.85E−07 25 4.39E−0920x 1.42E−08 15a 4.75E−08 40b 1.86E−07  6b 4.40E−09 11b 1.47E−08 20t5.41E−08 38a 1.87E−07  3i 5.53E−09  3a 1.57E−08 20r 6.15E−08 46a1.94E−07  6a 5.62E−09 36a 1.75E−08 20l 6.62E−08 50b 1.94E−07 23 5.67E−0920a 1.87E−08 20n 7.54E−08 45a 2.03E−07 20e 6.09E−09 13c 1.96E−08 20p7.64E−08 34 2.89E−07  3e 6.84E−09 13b 1.97E−08 46ad 7.96E−08 46ak2.96E−07 20m 7.34E−09 20aa 2.12E−08 46al 8.38E−08 52aa 3.43E−07  3d7.35E−09 20y 2.22E−08 20c 8.47E−08 38b 4.41E−07 14b 7.35E−09 20q2.78E−08 42b 9.25E−08 52e 4.42E−07 13a 8.18E−09 16b 2.83E−08 20s9.78E−08 45b 5.15E−07  3h 9.21E−09 17b 2.95E−08 31 1.11E−07 43b 5.65E−0714a 9.59E−09  3f 3.00E−08 20z 1.13E−07 52l 5.67E−07 20ah 9.85E−09 36e3.34E−08  9a 1.18E−07 52ac 6.59E−07 20d 1.07E−08 20f 3.41E−08 40af1.21E−07 35q 9.89E−07 20v 1.11E−08 20ab 3.51E−08 15b 1.27E−07 52b1.05E−06 20g 1.19E−08 20i 4.10E−08 48a 1.45E−07 35a 1.07E−06  3b1.30E−08 20k 4.55E−08 39b 1.55E−07 35e 1.18E−06 20ae 1.31E−08

To verify these findings, the MES program was utilized on the p50subunit of NF-κB to identify any potential binding areas for theanalogs. The results of this docking study are slightly different thanthose when the MES program was not used. All the potential inhibitorsbind to an area directly below the DNA binding area and wrap around tothe backside of the protein, as shown in FIG. 19, indicating they mayinhibit the NF-κB-DNA interaction. None of the potential inhibitors bindin the “small hole” as in the docking results without the MES program(FIG. 17). The K_(est) values for these analogs are mediocre, with thebest analog, 15a, having a K_(est) of 2.27E-08 M as shown in Table 9.Since the library of analogs does not display good K_(est) values orcorrelate with any K_(exp) results, NF-κB does not appear to be thetarget for curcumin analogs.

TABLE 9 K_(est) Values for NF-κB (1SVC) with MES. 15a 2.27E−08 11b4.15E−07 20l 8.98E−07 20ac 2.12E−06 17b 2.48E−08  3f 4.22E−07  3b9.38E−07 40b 2.13E−06 12b 2.63E−08 40af 4.26E−07 20d 1.01E−06 48ad2.31E−06 20ag 5.48E−08  3g 4.37E−07 20k 1.19E−06 52aa 2.40E−06 15b6.30E−08  3e 4.45E−07 52e 1.21E−06 31 2.95E−06 38a 7.76E−08 20ah4.51E−07 20e 1.22E−06 20s 2.98E−06  9a 8.10E−08  3a 4.79E−07 20f1.27E−06 39b 3.04E−06  6a 8.37E−08 20u 4.82E−07 20g 1.28E−06 46ak3.31E−06 53 9.53E−08 20ae 5.00E−07 20ab 1.34E−06 20t 3.52E−06  3h1.01E−07 13c 5.36E−07 20y 1.34E−06 36e 3.67E−06  3d 1.29E−07 20o5.47E−07 52l 1.46E−06 20b 4.27E−06  9b 1.32E−07 14b 5.50E−07 20aa1.47E−06 52ac 4.50E−06 23 1.58E−07 20w 5.71E−07 20x 1.55E−06 42b6.29E−06 25 2.36E−07 20a 5.80E−07 20z 1.58E−06 50b 6.32E−06  3i 2.67E−0720n 6.46E−07 20c 1.70E−06 52b 7.86E−06 14a 2.78E−07 46ad 6.58E−07 20q1.74E−06 35q 8.52E−06 20p 3.01E−07 36a 6.79E−07 20r 1.77E−06 45b9.20E−06 13a 3.13E−07 46a 6.81E−07 46al 1.81E−06 35a 1.11E−05 20i3.22E−07 13b 7.62E−07 29 1.95E−06 35e 1.30E−05 16b 3.31E−07  6b 7.66E−0738b 2.02E−06 34 1.84E−05 20m 3.58E−07 45a 8.30E−07 48a 2.06E−06 43b1.88E−05 20v 4.08E−07

Example 5 Inhibition of AP-1 Activity by Curcumin Derivatives

Curcumin and its analogues were screened for activity against AP-1 by acellular assay using the AP-1 stable cell line (293/AP1-luc). The cellline is derived from human 293 embryonic kidney cells containing achromosomal integration of a luciferase reporter construct regulated by3 copies of the AP-1 response element (Panomics, Inc.). This cell lineis obtained by co-transfection of pAP1-luc and pTK-hyg containingplasmids followed by the addition of hygromycin (200 μg/ml) to maintaincell selection.

The cell line was grown in a humidified atmosphere at 37° C. in 5%CO₂/95% air and maintained in Dulbecco's Modified Eagle's Medium(DMEM-high glucose containing 4 mM glutamine) containing fetal bovineserum (FBS, 10%), sodium pyruvate (1 mM), penicillin (100 units/ml),streptomycin (100 μg/ml) and hygromycin (100 μg/ml) to maintain cellselection (Gibco/Invitrogen).

The 293/AP1-luc cells were re-plated, 24 hr prior to treatment into,24-well cell culture plates in media without hygromycin, to prevent itfrom interfering with the assay. The cells were then allowed to grow andattach, to the wells, for 24 hr in a humidified atmosphere at 37° C. in5% CO₂/95% air. After 24 hr, the cells had reached approximately 60%confluency. The cells were then given media (1 ml) with or withoutphorbol 12-myristate 13-acetate (PMA, 10 ng/ml, Calciochem) followed byimmediate treatments with curcumin or analogue (15 μM in DMSO). Thecells were placed again in a humidified atmosphere at 37° C. in 5%CO₂/95% air for 24 hr. Plate wells were gently washed with PBS, pH 7.4,and lysed with passive lysis buffer (1x, 100 μl, Promega). Thesubsequent chemiluminescent lysates were analyzed with the LuciferaseAssay System (Promega) utilizing a TD-20/20 luminometer. The relativelight units (photons) were determined by the addition of fireflyluciferase substrate (75 μl) to cell lysate (10 μl). The light unitswere then normalized to the amount of protein in the well (mg/ml) withBCA™ Protein Assay Kit (Pierce) and standardized to percent of control(PMA).

To determine cell viability, cells were treated as above but with 15 μManalogue. After gently washing to remove any dead cells, they were givenmedia (100 μl) and CellTiter 96® AQueous One Solution reagent (20 μl)for 1 hour and read at 490 nm with a Spectromax plate reader.

Curcumin is a known inhibitor of the AP-1 activation cascade. Therefore,modification of the structure of curcumin could lead to analogs withenhanced activity. The library consisting of three series of curcuminanalogs were used to examine the role of the enone functionality in arylsystems where the spacer is 7-carbons (as in curcumin), 5-carbons or3-carbons in length. In addition, the importance of aryl ringsubstituents was assessed. The AP-1 activities of curcumin and analogswere determined by a cellular firefly luciferase assay. This assayutilized a commercially available cell line (Panomics 293-luc cellularassay) developed for screening inhibitors of AP-1. This cell line isstably transfected with a luciferase reporter controlled by an AP-1dependent promoter. The cell is stimulated with phorbol ester whichactivates AP-1. AP-1 then binds to one of three promoter regions on thecells DNA leading to the production of a luciferase enzyme. Luciferin isadded to the cell lysates and the luciferase enzyme catalyzes a cleavageof luciferin leading to the emission of light.

FIGS. 20A-C show analogs active in the AP-1 cellular assay. The activeanalogs in FIGS. 20A-C are arranged from highly active on the left toslightly active on the right. Figures containing all analogs can befound in FIGS. 21A-C.

Active analogs in series 1, which contain a 7-carbon spacer, are shownin FIG. 20A. Two analogs, 6a and 9a, in this series were more activethan curcumin (3a). Both of these analogs contain the same aryl ringsubstituents as curcumin in addition to either a methyl (6a) or benzyl(9a) substituent on the central methylene carbon. A third active analog,9b, also contains a central methylene benzyl substituent. No activeanalogs in this series contained a saturated spacer between the arylgroups. Four of the seven analogs in this series display activity inboth antioxidant assays. It is important to note that three analogs wereactive against AP-1 independent of antioxidant activity.

Active analogs in series 2, which contain a 5-carbon spacer, are shownin FIG. 20B. Eleven analogs, 20m, 20ag, 31, 20c, 20w, 29, 38a, 20l, 20o,20q and 20d, in this series were more active than curcumin. Of theseeleven active analogs, nine contain substituted aryl groups. Six analogscontain substituents ortho to the spacer on the aryl group, indicatingthis position may be important for AP-1 activity. Analogs 29 and 31contain pyridine rings with no substituents on the ring. These twoactive analogs indicate that if the analogs in this series have aspecific target, the target may contain a hydrogen bond donor in thearea of binding. Since only three of the eleven active analogs in thisseries display antioxidant activities, it is suggested that theseanalogs are targeting a specific protein.

Active analogs in series 3, which contain a 3-carbon spacer, are shownin FIG. 20C. No analog in this series was more active than curcumin. Theactive analogs in this series also exhibit good antioxidant activities.The two most active analogs, 45a and 48a, in this series displayedantioxidant activity in both antioxidant assays. Active analogs, 35q,46ad and 46al, in this series were also active in one or the otherantioxidant assay.

The IC₅₀ values for the twelve active analogs as well as curcuminagainst AP-1 were also measured. IC₅₀ plots for these active analogs areshown in FIGS. 22A-L. Of the twelve best analogs against AP-1, nine ofthe analogs also ranked in the top twelve against NF-κB activity. Table10 shows the IC₅₀ values of the nine analogs that were active againstboth NF-κB and AP-1. Table 10 also shows whether each analog was activeas an antioxidant (+) in both the TRAP and FRAP assays.

TABLE 10 IC₅₀ Values and Antioxidant Results for Active Analogs AgainstNF-κB and AP-1. NF- AP-1 κB Analog IC₅₀ IC₅₀ Structure Number (μM) (μM)TRAP FRAP

20m 1.4 6.4 − −

31 4.1 3.4 − −

9a 5.3 7.6 + +

6a 6.0 6.7 + +

38a 7.3 4.2 + +

29 8.2 3.5 − −

20ag 8.3 5.4 − +

20q 11.7 4.2 + −

3a 12.8 8.2 + +Of the IC₅₀ values obtained, curcumin (12.8 μM) is the least potentanalog against AP-1. Analog 20m which has an ortho substituent is themost active analog against AP-1 with an IC50 value of 1.4 μM. Asobserved in Table 10, several analogs are active against AP-1independent of antioxidant activity. This indicates that the analogs aretargeting specific proteins in the cell. Since nine of the twelve bestanalogs against AP-1 are also active against NF-κB it is possible thatthese analogs are acting on a common target involved in both activationcascades and that the analogs are not inhibiting the AP-1 or NF-κBproteins directly.

Example 6 Molecular Modeling of the Binding of Curcumin Derivatives toAP-1

When performing docking studies of the potential inhibitors againstAP-1, one crystal structure (1FOS) was selected from the twenty fiveselections that were available. 1FOS (Glover et al., Nature 373, 257-261(1995)) was selected because it contained the c-Jun and c-Fosheterodimer, the most common heterodimer, complexed to a segment of DNA.This crystal structure was important because it contained the exactbinding site of DNA to this heterodimer. This provided informationconcerning the blocking of AP-1-DNA binding interactions by the analogs.

FIG. 23 shows the c-Jun and c-Fos AP-1 heterodimer bound to a segment ofDNA. In FIG. 23, the blue protein is the c-Jun/c-Fos heterodimer and theyellow segment is the DNA. When the DNA is removed as shown in FIG. 24,a “Y” shaped area is exposed. It is in this location that the analogswill bind if they are preventing an AP-1-DNA binding interaction. Whendocking studies were performed, the potential inhibitors bound in theentire DNA interaction region. The front side of these bindinginteractions is shown in FIG. 25 and the backside of these bindinginteractions is shown in FIG. 26.

The analogs that appear to be coming over the top in FIG. 26 are thesame analogs as in FIG. 25. Most of these analogs bind in the exactregion as the DNA was bound. However, the analogs have mediocre K_(est)values with analog 9b displaying the best inhibition with a K_(est) of6.53E-8 M as shown in Table 11. There is no correlation to the K_(exp)results. This indicates that the analogs do not bind to the c-Jun andC-Fos heterodimer or at the very least, they do not inhibit DNA frombinding to AP-1.

To verify these findings, the MES program was utilized on the AP-1heterodimer to identify any potential binding areas for the potentialinhibitors. The results of this docking are similar to those from whenthe MES program was not used (FIG. 27). All of the analogs still bind tothe area directly below the DNA binding area and indicate a possibleinhibition of AP-1-DNA binding interactions. Once again, the potentialinhibitors have mediocre K_(est) values with analog 15a having a K_(est)value of 1.26E-7 M as shown in Table 12. However, there is nocorrelation to the K_(exp) results which indicates that the analogs donot bind to the c-Jun and C-Fos heterodimer or at the very least, theydo not inhibit DNA from binding to AP-1.

TABLE 11 K_(est) Values for AP-1 (1FOS).  9b 6.53E−08 38a 7.53E−07 20u2.07E−06 20r 4.30E−06 12b 9.96E−08  3h 7.73E−07 20ah 2.13E−06 13b4.60E−06 15a 1.57E−07 20z 8.46E−07 52l 2.14E−06 20p 4.90E−06  3d1.71E−07 20w 8.98E−07 20t 2.35E−06 45a 5.01E−06  6a 1.73E−07 20k9.23E−07 11b 2.45E−06 20x 5.14E−06 53 1.95E−07 20ac 9.70E−07 36a2.54E−06 46ak 5.25E−06 46ad 2.37E−07 20g 1.07E−06 20c 2.62E−06 20e5.33E−06 20m 2.43E−07  6b 1.16E−06 20aa 2.72E−06 39b 5.88E−06 232.44E−07 20ag 1.20E−06 20ab 2.76E−06 46a 6.66E−06 20d 2.88E−07 20l1.28E−06 16b 2.82E−06 52b 6.83E−06 20v 2.99E−07 17b 1.28E−06 52e2.92E−06 42b 6.84E−06 48ad 4.18E−07  3e 1.43E−06 20b 2.93E−06 20f7.63E−06 13c 4.38E−07 20ae 1.53E−06 52aa 2.96E−06 50b 8.05E−06  9a4.49E−07 20y 1.60E−06 29 3.12E−06 48a 1.10E−05  3a 5.01E−07 20q 1.60E−0640af 3.24E−06 34 1.11E−05 25 5.31E−07 13a 1.75E−06 20s 3.30E−06 45b1.19E−05 15b 5.38E−07 36e 1.83E−06 31 3.38E−06 43b 1.91E−05  3i 5.42E−0752ac 1.85E−06 20n 3.57E−06 40b 1.99E−05 20i 5.49E−07 14b 1.86E−06 20a3.93E−06 35e 5.48E−05  3g 6.19E−07  3b 1.97E−06 38b 4.02E−06 35a5.53E−05 14a 6.61E−07  3f 1.99E−06 46al 4.17E−06 35q 1.02E−04 20o7.31E−07

TABLE 12 K_(est) Values for AP-1 (1FOS) with MES. 15a 1.26E−07 20g1.38E−06 52e 2.79E−06 20e 5.33E−06 13c 2.05E−07 17b 1.49E−06 20y2.93E−06 20q 5.40E−06 23 2.96E−07  3i 1.53E−06  3f 2.99E−06 52b 6.00E−0646ad 3.15E−07  3d 1.57E−06 20k 3.28E−06 46ak 6.04E−06 53 3.43E−07  3h1.59E−06 20aa 3.29E−06 20b 6.33E−06  3g 4.75E−07 13b 1.74E−06 20x3.33E−06 20p 6.38E−06 20d 5.74E−07 12b 1.79E−06 20ah 3.40E−06 38b6.95E−06  9b 6.11E−07  6a 1.83E−06 11b 3.58E−06 20t 8.29E−06 25 6.78E−0720s 1.86E−06 20ac 3.67E−06 34 8.51E−06 20z 7.14E−07 52aa 1.98E−06 46a3.76E−06 20f 8.58E−06 20v 9.80E−07  6b 2.02E−06 14b 3.78E−06 40b8.61E−06 38a 1.04E−06  3a 2.03E−06 36e 3.79E−06 36a 9.23E−06 48ad1.08E−06 14a 2.04E−06 31 3.81E−06 20r 9.23E−06  9a 1.12E−06 20ae2.07E−06 20i 3.86E−06 48a 1.04E−05 20w 1.20E−06 52ac 2.13E−06 52l3.95E−06 50b 1.07E−05 20ab 1.22E−06 20m 2.17E−06  3b 4.00E−06 43b1.09E−05 15b 1.22E−06 13a 2.34E−06 29 4.08E−06 45b 1.45E−05 20ag1.24E−06 42b 2.58E−06 20a 4.26E−06 45a 1.78E−05 20o 1.26E−06 20n2.60E−06 20l 4.65E−06 35a 4.51E−05  3e 1.29E−06 46al 2.74E−06 40af4.80E−06 35e 4.90E−05 20u 1.29E−06 20c 2.76E−06 39b 4.97E−06 35q1.10E−04 16b 1.30E−06

Example 7 Evaluation of Curcumin Derivative Pharmacophores Using QSAR

A QSAR analysis of the data was carried out using the Catalyst program(Accelrys). A wide range of structures and activities from the resultsdescribed for FIGS. 3-5 were used to generate multiple pharamcophores. Asingle pharmacophore did not provide a satisfactory fit of the data.Moreover, pharmacophores that were derived separately from 5-carbonanalogs or from 3-carbon analogs did not provide satisfactory fits.However, a single pharmacophore could provide a satisfactory fit of thedata for analogs in the 7-carbon series. FIG. 28 shows a pharmacophoreon which curcumin is superimposed. In FIG. 28, Curcumin was aligned withthe pharmacophore model generated with the Catalyst program, usingcompounds 3a, 3e, 6a, 9a, 12b, 14a, and 14b as the training set. Thepharmacophore model consists of two hydrophobic aromatic regions withcenters 11.8 angstroms (Å) apart and a hydrogen bond acceptor 6.2 Å fromthe nearest hydrophogic aromatic region and 7 Å from the other. Thepharmacophore provided an excellent fit (correlation 0.9) of analogs onthe 7-carbon series. The inability of a single pharmacophore to providea satisfactory fit of all of the data or of the data from the 3-carbonor 5-carbon series may mean that there are several different targets forthese analogs.

Example 8 Reactivity and Bioavailability of Curcumin Derivatives

Curcumin is considered a very non-toxic compound but with limitedbioavailability. D Ranjan et al., (2004) J Surg Res 121, 171-177. From amedicinal chemistry perspective, a potential concern regarding thestructure of curcumin and its analogs is the presence of one or twoα,β-unsaturated ketone functional groups. Weber et al., (2005) BioorgMed Chem 13, 3811-3820. These groups potentially serve as Michaelacceptors which are chemically reactive and can form undesirablecovalent modifications with biomolecules. Michael acceptors aregenerally thought to be poor drug compounds because of their highreactivity toward nucleophilies. In order to evaluate the potential ofcurcumin as a Michael acceptor, its reactivity toward L-cysteine as thenucleophile donor was examined. Curcumin (20 μM) was incubated with 1 mML-cysteine in 0.1 M sodium phosphate buffer, pH 7.0, and measuredcurcumin's spectral properties at 425 nm as a function of time.L-Cysteine rapidly reacted with curcumin and quenched absorbance with at_(1/2) of 7.2 min., as shown in FIG. 29. Thus, curcumin is highlyreactive towards nucleophilies; this may explain the poor oralbioavailability of curcumin but also suggests that its toxicity mayappear to be deceptively low owing to reactions of curcumin with dietarycontents.

The high reactivity of curcumin, presumably through Michael addition tothe α,β-unsaturated ketone functionality, and the fact that thoseanalogs that are more active than curcumin are generally α,β-unsaturatedketones raised the question whether any analogs devoid of thisfunctionality retain activity. Screening of the prepared curcuminderivatives identified two analogs (42b and 52b) that show activitycomparable to curcumin. Neither 42b nor 52b retain the α,β-unsaturatedketone functionality. This suggests that analogs that are much lessreactive than curcumin can be developed as inhibitors of NFκB.

Non-specific inhibition of protein drug targets by small moleculeinhibitors can arise by formation of large molecular weight aggregatesin an aqueous environment, which provide a microenvironment for proteinadsorption that can produce apparent but false inhibition. McGovern etal., (2002) J Med Chem 45, 1712-1722. For example, compounds withmulti-aryl ring structures have a tendency to assemble into highlyordered complexes driven by the stacking of their aromatic rings.Compounds that aggregate may have little potential as lead compounds fordrug development. Accordingly, whether curcumin aggregates in an aqueousenvironment was evaluated by measuring the potential aggregation ofcurcumin using a standard light scattering assay. Curcumin was dilutedto 1.0-20.0 uM in 0.1 M phosphate buffered saline, pH 7.0, and itsabsorbance was measured at 425 nm. The absorbance profile remainedlinear throughout the concentration range tested, suggesting noaggregation.

Summary of Observations Regarding Activity of Curcumin Derivatives

Curcumin has a broad range of biological activities, some of which mayderive from its anti-oxidant activity or ability to quench free radicalreactions and some that involve inhibition or inactivation of specifictargets. Curcumin can scavenge superoxide radicals, hydrogen peroxideand nitric oxide, and it has been suggested that the ability of curcuminto protect against radiation damage, iron-induced hepatic damage,xanthine oxidase injury and oxidative stress depends upon theanti-oxidant and free radical-scavenging properties of curcumin (Joe etal., Crit. Rev. Food Sci. Nutr. 44, 97 (2004); Bonte et al., Planta Med.63, 265 (1997); Reddy et al., Toxicology 107, 39 (1996); Cohly et al.,Free Radical Biol. Med. 24, 49 (1998)).

Study of the inhibition of activation of NF-κB by analogs of curcumindemonstrated that a) some analogs are more active than curcumin; b) notall analogs that are active need retain the enone functionality, andthus there is reason to expect that some active curcumin derivatives maybe much less reactive than curcumin; and c) analogs with heterocyclicrings are active. Furthermore, there appear to be several differenttargets that are involved in prevention the activation of NF-κB byanalogs of curcumin. Also, the anti-oxidant activity of curcumin andanalogs is not required for activity against activation of NF-κB.

In Example 2, the abilities of curcumin and derivatives to quench thepre-formed radical monocation of2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), known as theTotal Radical-trapping Anti-oxidant Parameter (TRAP) assay, and theabilities of these compounds to reduce the ferric tripyridyltriazinecomplex, known as the Ferric Reducing/Anti-oxidant Power (FRAP) assay,were demonstrated (Schlesier et al., Free Radical Res. 36, 177 (2002)).It is noteworthy that many of the most active derivatives with regard toNF-κB show no activity in the TRAP or FRAP assay, which leads to theconclusion that there is no correlation between anti-oxidant activityand ability to inhibit the TNFα-induced activation of NF-κB. While notintending to be bound by theory, the lack of correlation between theanti-oxidant activities of curcumin and derivatives and the abilities ofthese compounds to inhibit the TNFα-induced activation of NF-κB and thePMA-induced activation of AP-1 suggests that curcumin and itsderivatives inhibit a specific target (or targets) rather than functionthrough general redox chemistry.

In summary, derivatives of curcumin in which the two aryl rings areseparated by 7-carbon, 5-carbon or 3-carbon spacers are able to inhibitthe TNFα-induced activation of AP-1 or NF-κB. However, activities canvary widely. The most active derivatives retain the enone functionality,although this functionality is not essential for activity. In addition,derivatives with the 5-carbon spacer are generally the most active. Ringsubstituents are not necessary but can affect activity. In addition, thearyl rings can be nitrogen heterocycles. The inhibition of TNFα-inducedactivation of NF-κB by curcumin and derivatives may occur at the levelof the IKK complex.

Example 9 NFκB as a Target for Alzheimer Drugs

New lead compounds as inhibitors of NF-κB will be identified largelybased upon the experimental data we will obtain by testing our libraryof curcumin analogs. We will apply quantitative structure-activityrelationship (QSAR) and ligand-based virtual screening (LBVS)technologies, using the available active/rigid hits, to develop apharnacophore model. At the same time, we will use 2D-fingerprints and3D properties (e.g., shape, electrostatics, pharmacophore fingerprints)to query the iResearch™ and ChemDiv libraries for additional putativeinhibitors of NF-κB. We have access to over 14 million unique structuresthat are available for synthesis, from the ChemNavigator(http://www.chemnavigator.com) iResearch™ Library.

The methodology for database preparation, in view of LBVS technologies,has been described in detail. Zamora et al., (2003) J Med Chem 46,25-33. Briefly, the strategies for compound selection include thefollowing steps: 1. database assembly (‘in silico’ inventory); 2a.structural integrity verification (keep unique structures only); 2b.limited exploration of alternative chemical representations for uniquestructures (stereoisomers, tautomers, ionization states); 3. propertyand structural filtering (remove unwanted structures); 4. 3D-structuregeneration (for virtual screening or 3D-based similarity); 5a.clustering or statistical design for selection; 5b. similarity-basedselection (if bioactives are known); 5c. receptor-based selection (iftarget binding site is known); 6. add a random subset to the final list.We have developed extensive knowledge in handling large chemicaldatasets—in particular the iResearch™ library has over 13 million uniquestructures, whereas ChemDiv has “only” 0.5 million. We will query bothdatabases, using the most active and rigid analogs that we identify, toselect a small subset (up to 300 structures) for acquisition.

One of the potential issues related to curcumin, as discussed above, isthe presence of two α,β-unsaturated ketone functionalities. Michaelacceptors are often considered reactive species that can lead to falsehits under biochemical assay conditions. We anticipate that, by usingvarious LBVS methodologies, in particular 3D-similarity andpharmacophore queries, and by having an extensive library of curcuminanalogs that include members without this functionality, we will be ableto move beyond this particular moiety. There is a list of over 200substructural filters that we routinely use when selecting compounds foracquisition. Waller et al., (1993) J Med Chem 36, 4152-4160. This listis used by Discovery Partners International, in support of the MolecularLibraries Initiative of the NIH Roadmap for the compound acquisitionprogram. We base this effort on the chemical similarity principle, whichstates that ligands with similar features are expected to have similarbiologic activity. Rishton (1997). Drug Discov. Today 2, 382-338. It isexpressed as a number that quantifies the “distance” between a pair ofcompounds (dissimilarity, or 1 minus similarity), or how related the twocompounds are (similarity). By definition, similarity needs a reference:That of a chemical descriptor system (a metric by which similarity isjudged), as well as that of an object or class of objects—we need areference point to which objects can be compared. Similarity depends onthe choice of molecular descriptors (Olah et al., (2004) J Comput AidedMol Des 18, 437-449), the choice of the weighting scheme(s) and thesimilarity coefficient itself. The coefficient is typically based onTanimoto's symmetric distance-between-patterns (Tanimoto (1961) Trans NYAcad Sci 23, 576), and on Tversky's asymmetric contrast model. Multipletypes of methods are available for evaluation of chemical similarity.See P Willett, 1987, “Similarity and Clustering Techniques in ChemicalInformation Systems” Letchworth: Research Studies Press; and P Willett(2000) Curr Opin Biotechnol 11, 85-88.

Our approach is to develop composite similarity rankings based ondifferent similarity scores and descriptors. We start with 2D similaritybased on the 320-bit MDL keys (Martin (2001) J Comb Chem 3, 231-250), asimplemented in software from Mesa Analytics and Computing LLC(http://www.mesaac.com), to which we add the 3D similarity defined bythe shape and electrostatics properties derived from ROCS (Rapid Overlayof Chemical Structures (Durant et al., (2002) J Chem Inf Comput Sci 42,1273-1280), as implemented in software from OpenEye Scientific Software(http://www.eyesopen.com). An additional metric for 3D similarity willbe defined by the ALMOND pharmacophore fingerprints; these are generatedusing software from Molecular Discovery Ltd. (http://moldiscovery.com).For the general subset that matches (to some degree) the above 2D and 3Dcriteria, we will also use the pharmacophore query to match thecurcumin-related molecules using Catalyst™, part of the Accelrys system(http://www.accelrys.com). For 3D structure generation we will use theOMEGA software from Openeye. The overall result will be a small subsetof compounds (up to 300), to be acquired and tested as described herein.It is worth noting that, using 17β-estradiol as a query, the Oprea grouphas performed LBVS searches on a set of 10,000 molecules followingexactly the same methodology (i.e., composite similarity ranking using acombination of 2D- and 3D-similarity measures). The top scoring 100compounds were tested on GPR30 (Revankar et al., (2005) Science 307,1625-1630), a newly discovered G-protein coupled receptor that binds17β-estradiol with high affinity, and on the estrogen receptor alpha(ERα); by testing these 100 non-steroidal molecules, Oprea and coworkerswere able to identify selective nanomolar ligands for both GPR30(K_(i)=13 nM, 1 agonist), and ERα (K_(i)<8 nM, 2 antagonists). Bologa etal., (2005) Nature Chem Biol, submitted; Revanka et al., 2005, Science307:1625-1630).

The following section describes tests of the ability of compoundsdeveloped to prevent activation of NFκB and thereby inhibit theup-regulation of pro-inflammatory genes in microglial cells.

Following injury or infection, microglial cells become activated andrespond by the release of cytokines, which in turn initiate theinflammatory event. IL1 plays a central role in the inflammatory processand is produced in greatest quantity by microglia. As mentioned above,IL1 is known to affect the expression of over 90 genes including thosefor cytokines, cytokine receptors, tissue remodeling enzymes andadhesion molecules (O'Neill (1995) Biochim Biophys Acta 1266, 31-44),and both IL1 up-regulation in microglia as well as the IL1 responseinvolve signaling through NF-κB. We will use microglia cell line BV2 totest the curcumin derivatives. We will incubate BV2 cultured microglialcells with the test compound and will monitor the expression of IL6,which is regulated by NF-κB. Pinteaux et al., (2002) J Neurochem 83,754-763. We have chosen BV2 as our model glial cell because this cellhas recently been used to demonstrate curcumin-mediated inhibition ofNF-κB activity. Kang et al., (2004) J Pharmacol Sci 94, 325-328.Importantly, this study establishes that the BV2 glial cell linemaintains the capacity for NF-κB activation and sensitivity to curcumintreatment, thus providing us with an excellent cell culture model systemto improve upon the effectiveness of curcumin therapy with our analoglibraries. In addition, the BV2 cell line has been immortalized andexhibits phenotypic and functional properties of reactive microglia.Bocchini et al., (1992) J Neurosci Res 31,616-621. We will incubate BV2cells with varying concentrations of curcumin analogs and quantitate IL6gene expression by real-time PCR as a measure of NFκB inhibition. IL6 isa good reporter for NF-κB activity in glia since it shows almost nodetectable expression in resting glial cells, yet after stimulation withlipopolysaccharide (LPS) its expression markedly increases (Kang et al.,(2004) J Pharmacol Sci 94, 325-328) allowing us to obtain a clear,quantitative evaluation of the effects of our library of analogs. Wewill separately monitor the effects of the analogs on inhibition of theactivation of NF-κB to demonstrate that the effect of a given analog onNF-κB activity correlates with its effect on IL6, as would be predictedif IL6 expression depends upon NF-κB.

Murine BV2 cells are grown in DMEM supplemented with 10% fetal bovineserum, 100 units/ml penicillin, 100 ug/ml streptomycin at 37° C. in 5%CO₂/95% air. Cells will be plated in 96-well plates for assay (5×10⁴cells/well). When cells reach 80-90% confluency, they will be incubatedwith lipopolysaccharide (LPS, 0.2 ng/ml) (5) together with varyingconcentrations of curcumin and its analogs for 6 h at 37° C. 5% CO₂/95%air. Total RNA will then be extracted, isolated using an RNeasy kit(Invitrogen) and quantitated by measuring absorbance at 260 nm. One-stepreverse transcriptase (RT) coupled to real time PCR analysis will beperformed using an Applied Biosystems 7000 System. Primers (designed toamplify <150 bp) and TaqMan probe for IL6 transcript and NFκB transcriptwill be designed using Applied Biosystems Primer Express software.Primers and TaqMan Probe for β-actin will be used as an internalcontrol. Real-time PCR values obtained for β-actin will be used tonormalize values for IL6 and NF-κB expression to correct for loading orcell number differences between wells. Cycling parameters will bedetermined to optimize IL6, NF-κB and β-actin amplifications. Ourstarting parameters have been successful for amplification of manydifferent genes currently under study: 50° C. 10 min (RT reaction), 94°C. 2 min (RT enzyme inactivation, Taq Polymerase activation), 40 cycles92° C. 30 s, 60° C. 30 s, 72° C. 30 s. The 96-well plate format willpermit a high efficiency and rapid screen to accurately assessindividual analogs as well as obtain quantitative data to determineindividual Ki values. The Applied Biosystems 7000 System is capable ofmultiplexing 96 samples simultaneously in approximately 2 h. Toquantitate the effects of analogs on IL6 and NF-κB expression, we willuse the comparative C_(T) method. The amount of target message (IL6 andNF-κB in activated microglial cells with analog incubation) will benormalized to the internal reference (β-actin) and compared to thecalibrator (IL6 or NF-κB in activated microglial cells without analogtreatment). Since curcumin is an established inhibitor of NF-κBactivity, it will serve as our positive control in all experiments.

As an alternative, the Panomics TransBinding NF-κB assay kit will beused. This kit is designed for rapid and sensitive quantization of NF-κBp50 in nuclear extracts of control and treated cells. The ELISA-basedkit utilizes oligonucleotide with a consensus NF-κB binding site thathas been immobilized on 96-well plates. Complex is detected withantibody to p50 by use of HRP-conjugated second antibody andcolorimetric detection. The assay is much more sensitive and rapid thanEMSA.

Developing a Reporter Assay in a Format for High Throughput Screening(HTS)

We have already described our directed approach to design of drugs forAlzheimer's disease where curcumin is the starting lead compound for asynthesis/screening approach combined with virtual data base screeningto identify improved lead compounds. Here, we describe a HTS approachthat is not directed, at least initially, by availability of leadcompounds but rather is a mass screening approach. This will bedeveloped as a separate approach that will parallel the directedapproach. HTS approaches that are part of the MLSCN of the NIH RoadmapInitiatives require development of a screening assay and then approvalfor inclusion of the target and assay into the MLSCN.

The brute force approach of screening vast numbers of diverse chemicaleffectors, which is characteristic of HTS, is often beyond the resourcesof academic research; however, if the size of a chemical library is toosmall, the identity of valuable structural details may be missed thatcould limit our QSAR approach. To circumvent these obstacles, we plan todevelop a HTS methodology that incorporates the fundamentals of flowcytometry. Flow cytometry is a sensitive and quantitative method formeasuring cell fluorescence and is easily adaptable for assessing theeffects of diverse compounds at the single cell level. The sensitivityof this method has been documented at concentrations of fluorescentmolecules of 10-100 picomoles. Edwards et al., (2004) Curr Op Chem Biol8, 392-398. For the University of New Mexico Flow Cytometry CoreFacility, it is routine to analyze thousands to tens-of-thousands ofparticles per second(http://hsc.unm.edu/som/research/flowcyt/hypercyt.shtml). In the past,the ability to use flow cytometry for analysis of multiple samples hasbeen hampered by the need for labor intensive sample handling. Recentgenerations of instrumentation have incorporated automated samplehandling to routinely process in excess of 1 sample/sec. The HyperCyt®high-throughput flow cytometry platform now used by our Core Facilityintegrates a flow cytometer with rapid autosampling which will allow usthe opportunity to develop a HTS protocol to screen a larger chemicallibrary than was possible in the past. This freedom will undoubtedlyenable us to obtain more structural information on NFκB inhibitors tomore rapidly advance our QSAR methodologies.

To accomplish this objective, we plan to re-engineer the 293T cells,used for the initial screening of curcumin derivatives, to express areporter system that includes four copies of the NF-κB DNA bindingsequence upstream from a green-fluorescent-protein (GFP) gene. We willthen use a lentiviral-based vector system that stably integrates intothe host genome. Buchschacher et al., (2000) Blood. 95, 2499-2504. TNFαwill provide the stimulus for activation of NF-kB as before and cellswill be simultaneously treated with varying amounts of curcumin and itsanalogs. TNFα treatment alone is expected to activate expression of GFPand will serve as our positive control. If curcumin or its analogs arecapable of preventing the TNFα-induced activation of NF-κB, we shouldsee quantitative reduction in fluorescence intensity due to diminishedGFP expression. Since analysis by flow cytometry is fully quantitative,we will be able to accurately titrate the effectiveness of each chemicaltoward NFkB inhibition. Analyzing the cells with the HyperCyt®high-throughput flow cytometry platform will allow us to process thisendpoint assay at rates of 20 to 40 samples/minute over a 4-fold rangeof fluorescence intensity.

To engineer a reporter cell line, we will obtain the lentivirus-basedreporter vector, pTRF1-NFkB-dscGFP, from System Biosciences (MountainView, Calif.) and develop a stably transfected 293T cell line. We planto use 293T cells since they are known to express NF-κB which is fullyresponsive to TNFα activation as demonstrated by their usefulness inluciferase-based assays. The lentivirus-based expression system containsall of the genetic elements that are necessary for packaging,transduction, stable integration of the viral expression sequence intogenomic DNA, and high level expression of the GFP reporter sequence thatis completely dependent on NFκB activation. To create a stablytransfected 293T cell line, the first step requires generation offunctional pseudoviral particles. To generated these particles, theexpression vector containing the NFkB binding domains and GFP reportersequence will be co-transfected with the packaging vector into apackaging cell line, typically 293T cells. The expression vector is thenreplicated intracellularly and packaged into pseudoviral particles,which contain the coat proteins necessary for delivery to mammaliancells. The pseudoviral particles will then be purified and used todeliver the reporter vector to freshly plated 293T cells. Oncedelivered, the viral reporter vector integrates into the host genome andexpresses the GFP gene following NFkB activation by TNFα. Upon stableintegration into the genome, the 5′ LTR promoter is inactivated, whichprevents replication of the viral sequence and formation of competentviral particles. Dull et al., (1998) J Virol 72, 8463-8471. Also, thetransduced cell no longer has the necessary genes to produce viralcapsid protein providing an additional safeguard. The efficiency oflentiviral transduction is close to 100% so this system should generatean ideal GFP-based NFkB reporter vector.

293T/NFκB-GFP cells will be plated into 96-well cell culture microtiterplates and, after 24 h, will be incubated with 20 ng/ml recombinant TNFαtogether with varying concentrations of curcumin and its analogs. Thecells will be incubated at 37° C. in 5% CO2/95% air for 7 hours. Flowcytometric analysis will be done as follows. Cells will be rinsed withphosphate buffered saline, pH 7.4, and detached by trypsin treatment.The 96-well microtiter plate will then be placed into the autosampler ofthe HyperCyt® system. The sampling probe then moves from one well to thenext drawing cells into the HyperCyt® system using a peristaltic pump.Between wells, the continuously running pump draws a bubble of air intothe sample line. This results in the generation of a tandem series ofbubble-separated samples for delivery to the flow cytometer with databeing collected in a single uninterrupted stream. The data are thenoptionally analyzed by proprietary software developed at the Universityof New Mexico by Dr. Bruce Edwards (Department of Pathology, School ofMedicine). With this format, one 96-well plate can be analyzed inapproximately 3 min thus achieving the speed necessary for a HTS assay.The procedures for development of the 293T/NF-κB-GFP cell and use ofthis cell in HTS are summarized in FIGS. 30 and 31.

Example 10 Effects of Curcumin Derivatives on Aβ Peptide Aggregation

The neuropathology that defines Alzheimer's disease has been wellstudied. One of the earliest histological changes seen in the brains ofAD patients is the deposition of amyloid-like plaques. Although manyblood-borne proteins have been identified in these plaques, the mainconstituent is a 40 or 42 amino acid hydrophobic peptide called Aβ(Glenner et al., 1984, Applied Pathology 2:357-369). Deposition ofplaques is thought to begin in the entorhinal complex and hippocampus,later progressing into the neocortex (Terry, 2000, Annals of Neurology47:421). The course of this disorder, which can last from months to wellover 10 years, is accompanied by a decrease in neural metabolic activityand increase in neural cell death. Clinically, patients suffer from avariety of unpredictable behaviors including loss in cognition, poorlearning and memory, and severe mood changes. The prevalence of thepathology increases from 3% of the population at age 65 to 47% after theage of 85 (Dyrks et al., 1993, FEBS Letters 335:89-93). Theamyloid-producing peptide, Aβ, is formed through proteolytic processingof the amyloid precursor protein (APP), a single pass transmembraneprotein that is cleaved by two independent aspartyl proteases (FIG. 32).

β-secretase, also knows as BACE (Vassar et al., 1999, Science286:735-741), cleaves in the ectodomain of APP proximal to the plasmamembrane (Cai et al., 2001, Nature Neuroscience 4:233-234). Followingβ-secretase cleavage, β-secretase performs a unique intramembraneproteolysis giving rise to the Aβ peptide, 40 or 42 amino acids inlength depending on β-secretase cleavage site choice within thetransmembrane spanning domain (Cai et al., 2001, Nature Neuroscience4:233-234; Selkoe, 2001, Physiological Reviews 81:741-766). Thisintramembrane cleavage event requires a multi-protein complex consistingof presenilin, nicastrin, Aph-1 and Pen-2, which together are thought toconstitute the β-secretase complex (Periz et al., 2004, J Neurosci Res77:309-322). Alternative to β-secretase cleavage, proteolysis can alsooccur by β-secretase, a member of the “A Disintegrin andMetalloproteinase-family” (ADAM) (Hooper et al., 2002, Curr Med Chem9:1107-1119). Cleavage by this enzyme, prior to the action ofβ-secretase, generates a non-amyloidogenic peptide approximately 25amino acids in length.

Although the Aβ peptide is continually produced in individuals of allages (Funato et al., 1998, Am J Pathol 152:1633-1640; Haass et al.,1992, Nature 359:322-325; Seubert et al., 1992, Nature 359:325-327;Shoji et al., 1992, Science 258:126-129), it has no known normalfunction. Since the formation of Aβ aggregates is largely dependent onthe concentration of available monomeric peptide, amyloid depositionrequires a shift in the balance of peptide degradation versusaccumulation (Glabe, 2000, Nat Med 6:133-134). Therefore, young, healthybrains are thought to have greater proteolytic catabolism of Aβ (Iwataet al., 2000, Nat Med 6:143-150) or, alternatively, increased endocyticclearance capacities for the peptide (Van Uden et al., 2000, Microsc ResTech 50:268-272; Van Uden et al., 2000, J Biol Chem 275:30525-30530).Abnormal accumulation of Aβ also appears to exert neurotoxic effects(Emre et al., 1992, Neurobiol Aging 13:553-559; Kowall et al., 1992,Neurobiol Aging 13:537-542; Rush et al., 1992, Neurobiol Aging13:591-594) by inducing oxidative stress (Yatin et al., 1999, NeurobiolAging 20:325-330; discussion 339-342) and loss of calcium homeostasis(Etcheberrigaray et al., 1998, Neurobiol Dis 5:37-45; Mattson et al.,1993, Trends Neurosci 16:409-414).

The Aβ fibrillogenesis hypothesis as the primary cause of AD has beenchallenged; however, the data suggesting that amyloid depositioncompromises synaptic integrity, along with memory and behavioralchanges, remains compelling (Hardy et al., 2002, Science 297:353-356;Soto, 1999, Mol Med Today 5:343-350). One of the first observationsnoted supporting this hypothesis was the localization of the gene forAPP on chromosome 21 (Goldgaber et al., 1987, Science 235:877-880). Onefacet of trisomy 21 (Down's Syndrome) consists of a gene dosage errorfor APP and results in an increase in Aβ amyloid burden and invariablyto the neuropathology of AD (Pallister et al., 1997, Neurobiol Aging18:97-103).

Moreover, a rare case of chromosomal translocation in Down's Syndromeinvolving chromosome 21 left the patient with a diploid copy of APPresulting in the absence of amyloid deposits and associatedneuropathology (Prasher et al., 1998, Ann Neurol 43:380-383). Additionalevidence supporting the Aβ hypothesis include; toxicity of Aβ fibrils tohippocampal and cortical neurons (Geula, 1998, Neurology 51:S18-29;discussion S65-67; Lorenzo et al., 1994, Proc Natl Acad Sci USA91:12243-12247), the natural occurrence of inherited mutations in APPresulting in increased Aβ formation sufficient to cause premature onsetof AD (Goate et al., 1991, Nature 349:704-706; Levy et al., 1990,Science 248:1124-1126), and mice transgenic for mutant human APPdemonstrate a time-dependent increase in Aβ production that directlycorrelates with the onset of neuropathological and behavioral changesassociated with AD (Games et al., 1995, Nature 373:523-527; Holcomb etal., 1999, Behav Genet 29:177-185; Hsia et al., 1999, Proc Natl Acad SciUSA 96:3228-3233; Hsiao, 1998, Exp Gerontol 33:883-889).

Once formed through β-/γ-secretase cleavage, the Aβ peptide is releasedfrom cells where it can aggregate and trigger events leading toneurotoxicity (Walsh et al., 2004, Neuron 44:181-193). NMR studies haveprovided evidence suggesting that the monomeric form of Aβ 1-40 peptideis initially unstructured and water-solvated between residues 1-14. Thisis followed by a long α-helical sequence encompassing residues 15-36 andincludes a hinge region around amino acids 25-27 (Coles et al., 1998,Biochemistry 37:11064-11077; Watson et al., 1998, Biochemistry37:12700-12706; Crescenzi et al., 2002, Eur J Biochem 269:5642-5648). Aβmost likely exists in a dynamic flux of different conformationsdepending on interactions with other molecules and metal ions (Maggio etal., 1995, Science 268:1920-1921; author reply 1921-1923). However, theunstructured peptide may represent a transitional conformation from anα-helix to β-sheet (Soto et al., 1995, Neurosci Lett 200:105-108). It iswidely believed that a conformational change in Aβ from an α-helix torandom coil to β-sheet configuration is largely responsible for itsextracellular aggregation and deposition (Wimley et al., 1998, Journalof Molecular Biology 277:1091-1110; Chen et al., 1997, FASEB Journal11:817-823; Gursky et al., 2000, Biochim Biophys Acta 1476:93-102; Roheret al., 2000, Biochim Biophys Acta 1502:31-43).

The initial stages of Aβ aggregation include oligomerization andprotofibril formation. Kinetic studies of Aβ fibrillogenesis havepointed to a nucleation-dependent mechanism for Aβ aggregation (Jarrettet al., 1992, Biochemistry 31:12345-12352; Jarrett et al., 1993, Cell73:1055-1058; Teplow, 1998, Amyloid 5:121-142). The rate of Aβoligomerization is markedly increased by “seeding” the reaction withother molecular components, such as apolipoprotein E or Aβ dimers, topromote peptide-peptide interactions (Evans et al., 1995, Proc Natl AcadSci USA 92:763-767). Based on NMR studies, Lansbury and coworkers haveproposed that two Aβ monomers dimerize through anti-parallelassociations after transitioning into their β-sheet conformations(Lansbury et al., 1995, Nat Struct Biol 2:990-998). Replacinghydrophobic amino acids within residues 17-21 of the Aβ peptideseriously impairs fibril formation, suggesting that anti-parallelassociations are stabilized by hydrophobic interactions, in addition tohydrogen bonding. Successive stacking of the anti-parallel structuresleads to the creation of a helical protofilament.

Lansbury and colleagues have envisioned Aβ aggregation as proceedingthrough four separate stages (Harper et al., 1997, Annu Rev Biochem66:385-407):

-   -   1. Protofibril formation requires >20 Aβ molecules,    -   2. protofibril elongation involves the reversible coalescence of        smaller protofibrils,    -   3. protofibril to fibril transition is the first pathogenic        feature of AD detected by dye binding, and    -   4. fibril elongation process is continuous.        Because the Aβ fibril was the first amyloid entity identified by        virtue of dye binding, it was naturally thought to represent the        form of Aβ peptide that was neurotoxic. However, more recent        evidence suggests that Aβ might be most detrimental to synaptic        integrity and neuronal health when it is in the form of soluble        oligomers generated during the early stages of the aggregation        pathway (Hartley et al., 1999, J Neurosci 19:8876-8884; Lambert        et al., 1998, Proc Natl Acad Sci USA 95:6448-6453; McLean et        al., 1999, Ann Neurol 46:860-866; Walsh et al., 2002, Nature        416:535-539; Walsh et al., 2004, Protein Pept Lett 11:213-228).        Microinjection of human Aβ into rats revealed that Aβ oligomers,        in the absence of monomers and amyloid fibrils, inhibit        long-term potentiation in the hippocampus (Walsh et al., 2002,        Nature 416:535-539). Therefore, because of the complexity of the        amyloid deposition process, inhibition of all forms of fibril        intermediates-including oligomers and protofibrils-should be        addressed when developing therapeutic intervention strategies.

Current therapy of Alzheimer's disease focuses largely on symptomaticaspects of the clinical pathology. Strategies include increasingcholinergic neurotransmission by administering acetylcholine esteraseinhibitors (e.g. Tacrine or Donepezil) (Mayeux et al., 1999, N Engl JMed 341:1670-1679), and more recently modulation of NMDA receptoractivity by Memantine (Reisberg et al., 2003, N Engl J Med348:1333-1341). However, although these therapies have shown a modesteffect on slowing cognitive decline, they have yet to demonstrate anymajor impact on the progression of the disease. Other targets in ADinclude the secretase enzymes. Logically, if one could reduce oreliminate activities of either β- or γ-secretase, then a concomitantreduction in Aβ accumulation would result. Since γ-secretase is anaspartyl protease, inhibitors have been designed to mimic the protease'stransition state. Benzodiazepine inhibitors have proven to be highlyeffective demonstrating IC₅₀ values as low as 0.3 nM (Seiffert et al.,2000, J Biol Chem 275:34086-34091). However, inhibition of γ-secretaseto treat AD is problematic because this protease has important functionsin normal physiologic processing of other critical substrates. Onesubstrate is the Notch receptor, which plays a vital role in cell fatedetermination during organismic development (Schweisguth, 2004, CurrBiol 14:R129-138). In fact, mice deficient in any of the γ-secretasesubunits (Presenilin-1, nicastrin, APH-1, or PEN-2) demonstrate a lethalphenotype due to the absence of Notch processing (De Strooper, 2003,Neuron 38:9-12).

Inhibition of β-secretase has also shown promise, but not withoutsignificant challenges (Citron, 2004, Trends Pharmacol Sci 25:92-97).Mice deficient in β-secretase are viable and show no Aβ peptidegeneration, suggesting that this aspartyl protease may be an attractivetarget for inhibitor design (Luo et al., 2001, Nat Neurosci 4:231-232;Roberds et al., 2001, Hum Mol Genet 10:1317-1324). Peptidic andpeptidomimetic inhibitors, mimicking transition state analogs, have beenfound (Turner et al., 2001, Biochemistry 40:10001-10006) oftendemonstrating IC₅₀ values in the nanomolar range (Turner et al., 2001,Biochemistry 40:10001-10006; Ghosh et al., 2001, J Med Chem44:2865-2868). However, although these peptide-based inhibitors havebecome important and necessary research tools, their low intrinsicstability and inability to cross the blood-brain barrier have hamperedstudies aimed at assessing their effectiveness in vivo (Citron, 2004,Trends Pharmacol Sci 25:92-97).

It is now well-established that Aβ deposition occurs as a result ofchanges in peptide structure. Following proteolytic cleavage, the Aβpeptide transitions from a soluble monomeric, α-helical structure to anelongated β-sheet conformer, thus exposing highly interactivehydrophobic amino acid residues. Recognizing the critical nature of thisconformational transition, efforts have been focused on developing leadinhibitor compounds that will stabilize the soluble form of Aβ byshifting the equilibrium from β-sheet to α-helix, since the soluble formis a better target for degradation and cellular clearance. Thus, themost promising potential therapies to date focus on compounds thateither prevent Aβ fibril formation or reverse the aggregation process,thereby reducing overall amyloid burden. The two most successfulapproaches toward reducing amyloid burden in vivo have utilized eitherantibodies specific for the Aβ peptide or non-peptidic small moleculeinhibitors. Elan Pharmaceuticals originally injected a transgenic mousemodel of AD with a combination of β-amyloid and an immune systemactivating agent. The injected mice demonstrated significant reductionsin amyloid plaques when compared to control animals (Schenk et al.,1999, Nature 400:173-177). Based on this finding, clinical trials werebegun using a synthetic form of the Aβ peptide, AN-1792. Unfortunately,Phase IIa trials were terminated when four patients developed symptomscharacteristic of encephalitis.

In drug development, non-peptidic small molecules have shown muchpromise toward Aβ peptide dissolution. These molecules are typicallyplanar, contain a scaffold of 2-4 phenyl rings and have a basic nitrogenon one of the rings. Examples include: Congo Red (Lorenzo et al., 1994,Proc Natl Acad Sci USA 91:12243-12247), naphtylazo derivatives of thedye Congo Red (Talaga, 2001, Mini Rev Med Chem 1:175-186), thewell-known antibiotic rifampicin (Tomiyama et al., 1994, Biochem BiophysRes Commun 204:76-83), anthracyclone derivatives (Talaga, 2001, Mini RevMed Chem 1:175-186), and benzofuran-based compounds (Allsop et al.,1998, Biochem Soc Trans 26:459-463). Although these molecules havedemonstrated low μM, and sometimes nM IC₅₀ values, they typically do notdisplay ideal drug qualities owing to their charged moieties; suchcharged groups enhance solubility properties, but restrict passageacross the blood-brain barrier (Scherrmann, 2002, Vascul Pharmacol38:349-354). In addition, many of these compounds have proven to behighly toxic.

Curcumin is a polyphenolic natural product from the spice turmeric,which comes from the root of Curcuma longa of the ginger family. Groundturmeric powder as it is used in culinary preparations contains 5%curcumin. The majority of the world's curcumin (80%) is produced andconsumed in India. In traditional Indian medicine, curcumin has beenused to treat a host of ailments through topical, oral and inhalationadministration, and has recently been found safe in six human trials atoral loads up to 8 grams/day for 6 months (Chainani-Wu, 2003, J AlternComplement Med 9:161-168). Most of the clinical trials of curcuminpertain to its anti-tumor activity in colon, skin, stomach, duodenal,soft palate and breast cancers. In addition, curcumin exhibitsanti-inflammatory activity and is a potent anti-oxidant and free radicalscavenger (Leu et al., 2002, Curr Med Chem Anti-Canc Agents 2:357-370).In APP-overexpressing transgenic mice, curcumin reduces levels ofoxidized proteins and inflammatory cytokine interleukin-1β (Lim et al.,2001, J Neurosci 21:8370-8377) thus offering a potential therapy againstmicroglial activation, which is routinely detected in brains from ADpatients. Curcumin has additional activities of interest: it limits theprogression of renal lesions in the STZ-diabetic rat model (Suresh etal., 1998, Mol Cell Biochem 181:87-96), and ameliorates oxidativestress-induced renal injury in mice (Okada et al., 2001, J Nutr131:2090-2095). Consequently, there has been extensive interest in theanti-oxidant properties of curcumin and the possibility that many of itsbiological activities are derived from its anti-oxidant properties(Balasubramanyam et al., 2003, J Biosci 28:715-721; Oyama et al., 1998,Eur J Pharmacol 360:65-71).

The large consumption of curcumin by the Indian population may helpexplain their relatively low (4 times less) incidence of AD compared tothe U.S. population (Chandra et al., 2001, Neurology 57:985-989).Although no systematic trials have been preformed using curcumin inIndia, recent studies have provided valuable insights on curcumin's rolein AD (Yang et al., 2005, J Biol Chem 280:5892-5901; Ono et al., 2004, JNeurosci Res 75:742-750). Curcumin was shown to inhibit the formation ofAβ oligomers and fibrils in vitro and reduce Aβ amyloid burden in vivo.The IC₅₀ for fibril inhibition is 0.8 μM and 1.0 μM for disaggregationof preformed fibrils. Importantly, curcumin administered as a dietarysupplement lowered Aβ deposition in aged APP(Swedish)-transgenic mice(Tg2576), clearly demonstrating its ability to cross the blood-brainbarrier in sufficient quantities to reduce amyloid burden. Curcumin isstructurally similar to other inhibitors of Aβ aggregation such as CongoRed and Chrysamine G, however replacing the charged moieties on theselatter compounds with polar groups greatly facilitates blood-brainbarrier passage (Klunk et al., 1994, Neurobiol Aging 15:691-698). Thisstudy establishes curcumin as one of the most promising lead compoundsin recent years that offers real potential for reducing amyloiddeposition in AD and in doing so halting or reversing diseaseprogression.

We hypothesize that the base structure of native curcumin provides anexcellent starting point to identify chemical analogs that have greateraffinity for Aβ peptide oligomers, greater efficiency in fibrildisaggregation and inhibition of fibril formation, while improvingbioavailability.

A key event in progression of Alzheimer's disease (AD) is aggregation ofthe Aβ peptide to form amyloid fibrillar deposits. A number of studieshave reported on inhibitors that are effective in preventing Aβaggregation. The usefulness of these inhibitors has been limited due totheir toxicity or their inability to cross the blood-brain barrier.However, it was recently reported that the natural product curcumin, anon-toxic component of the spice turmeric, not only prevents Aβaggregation but also disaggregates preformed Aβ fibrils. Curcumin wasreported to cross the blood-brain barrier when injected into thecirculation and to reduce amyloid plaque burden in vivo in a transgenicmouse model. Curcumin was less effective, however, when added to thediet, due to limited oral bioavailability. Although curcumin is clearlyable to disaggregate Aβ peptide fibrils in vitro, its effectiveness invivo has considerable room for improvement.

Based upon data presented in the recent literature, we hypothesize thatcurcumin presents molecular features that make it an excellent leadcompound for the development of more effective inhibitors of Aβaggregation that demonstrate improved IC₅₀ values while maintaining orimproving bioavailability.

In order to identify and improve upon the structural properties ofnative curcumin that are essential for its function in reversing amyloiddeposition, we have generated chemical analogs that include keymodifications to the base structure of curcumin. We plan to obtaindetailed information on the functional properties of our library ofcurcumin-based, chemical analogs toward Aβ peptide oligomerization. Weexpect to identify useful in vivo therapeutic inhibitors of amyloidplaque formation to halt or reverse cognitive decline.

First, we will identify the molecular features of curcumin that areresponsible for inhibition of Aβ peptide oligomerization using chemicalanalogs of curcumin. We have generated a chemical library of 84 novelcompounds based on the molecular structure of curcumin and will testtheir effectiveness in preventing Aβ peptide oligomerization anddisaggregating preformed fibrils. Using biotinylated Aβ (1-40) peptidein an electrophoretic mobility assay, we have obtained informativePreliminary Results examining the effects of select compounds from ourcurcumin-analog library on preventing Aβ oligomerization. We anticipatethat the results obtained will allow us to develop valuablestructure-activity relationships that will permit the identification ofnew compounds that will be effective for the prevention and/or treatmentof AD.

Next, we will determine whether analogs of curcumin are more effectivethan curcumin in reducing cytotoxicity caused by Aβ oligomers incultured neuronal cells. Depending on their concentration andaggregation state, Aβ oligomers are known to induce neurotoxic effects.We will determine if our curcumin-analogs are biologically active andprovide a neuroprotective effect against Aβ toxicity, using awell-characterized neuroblastoma cell line (SY5Y).

Finally, we expect to improve upon the efficacy of chemical analogs ofcurcumin using ligand-based drug design. Using the curcumin-analoglibrary, we will derive quantitative structure-activity relationship(QSAR) models based on experimental evidence obtained from the Aβoligomerization assays. We will select the most potent andconformationally rigid analogs to perform a ligand-based virtualscreening (LBVS) using several chemical libraries, focusing on the ˜0.5million compounds from Chemical Diversity (ChemDiv). The resulting LBVShits will be further tested with established in vitro Aβ oligomerizationassays and cell-based toxicity assays. We will seek to identifycandidates with both high potency and appropriate “drug-like” profile.

Curcumin reactivity: One potential concern regarding the structure ofcurcumin is the presence of two β-ketone-olefin moieties. These groupspotentially serve as Michael acceptors which are a chemically reactivespecies that can form undesirable covalent modifications with bindingtargets. These types of reactions can lead to false positives in invitro assays (Rishton, 1997, Drug Discov. Today 2:382-338). Michaelacceptors are generally thought to be poor drug compounds because oftheir high reactivity toward nucleophiles. In order to evaluatecurcumin's potential as a Michael acceptor, we examined its reactivitytoward L-cysteine as a nucleophile donor. We incubated 20 μM curcuminwith 1 mM L-cysteine in 0.1 M sodium phosphate buffer, pH 7.0, andmeasured curcumin's spectral properties at 425 nm as a function of time.L-Cysteine rapidly reacted with curcumin and quenched absorbance with at_(1/2) of 7.2 min (FIG. 33). Thus, curcumin is highly reactive tonucleophiles, providing a possible explanation for its poor uptake byintestinal epithelium. However, Aβ peptide contains no cysteine residuesso, although curcumin is highly reactive to this amino acid, itsinactivation by thiol nucleophiles should not pose a problem in ourinhibition studies. In addition, at or below neutral pH, primary aminogroups are predominantly in their protonated form and therefore weaknucleophiles that are unlikely to react to any significant extent withcurcumin.

Non-specific inhibition of drug targets by small molecule inhibitors canarise by their forming large molecular weight aggregates in an aqueousenvironment (McGovern et al., 2002, J Med Chem 45:1712-1722). Forexample, compounds with multi-aryl ring structures have a tendency toassemble into highly ordered complexes driven by the stacking of theiraromatic rings (Stopa et al., 1998, Biochimie 80:963-968; Stopa et al.,2003, Acta Biochim Pol 50:1213-1227). Since curcumin is a bi-phenolicmolecule, we investigated if it aggregates in an aqueous environment.Such an aggregation property would suggest that curcumin may show poorselectivity and thus have little potential as a lead compound foreffective amyloid dissolution in vivo. We measured potential aggregationof curcumin using a standard light scattering assay. Curcumin wasdiluted to 1.0-20.0 μM in 0.1 M phosphate buffered saline, pH 7.0, andits absorbance was measured at 425 nm. As shown in FIG. 34, theabsorbance profile remained linear throughout the concentration rangetested indicating no evidence of aggregation. From these data, weconclude that curcumin's ability to inhibit Aβ oligomerization is due tospecific interactions and not due to non-specific chemical aggregationeffects.

In the last two years, we have constructed a chemical library consistingof 84 curcumin-based analogs to identify the functional groupsresponsible for curcumin's established anti-oxidant properties (Barclayet al., 2000, Org Lett 2:2841-2843; Weber et al., 2005, Bioorg Med Chem13:3811-3820). The enone analogs include: 1) those that retain the7-carbon spacer between the aryl rings, 2) those with a 5-carbon spacer,and 3) those with a 3-carbon spacer. In addition to carbon spacervariations, analogs are also available with monoketone substitution forthe native diketone structure, varying degrees of saturation to test theimportance of unsaturation and addition of aryl rings to the unsaturatedcarbon spacer. We also have synthesized analogs that limit rotationalflexibility to address if stabilizing interactions with Aβ oligomerswill provide greater effectiveness in aggregate dissolution.

In order to perform large-scale screening of our analog library in arapid, reproducible and cost-effective manner, we are developing novelassays to accurately distinguish between monomeric and oligomeric Aβpeptide. We are in the process of developing an ELISA-based assay usingthe commercially available anti-Aβ oligomer specific antibody, A11(Glabe, 2004, Trends Biochem Sci 29:542-547; Kayed, 2003, Science300:486-489). This antibody clearly distinguishes the oligomericconformation of the Aβ aggregate, from the fibrillar or monomericpeptide. Since the oligomeric form of Aβ aggregates is receivingincreasing attention as a major factor responsible for synapticdysfunction (Hardy et al., 2002, Science 297:353-356), this antibodywill serve a very important diagnostic role due to its ability tospecifically recognize Aβ oligomeric conformers. We describe themethodology we are developing for this antibody in Research Plan.

However, as an initial screen of a select number of curcumin analogs fortheir ability to prevent Aβ aggregation in vitro, we have used thepublished SDS-PAGE mobility-shift procedure described by Yang, et al.,in their original identification of curcumin as an inhibitor of amyloidβ-oligomerization (Yang et al., 2005, J Biol Chem 280:5892-5901), with afew modifications. The original procedure relies upon resolvingcurcumin-treated and non-treated Aβ oligomers by SDS-PAGE, followed byimmunoblotting with an anti-Aβ peptide antibody. We have chosen a moredirect approach using biotinylated peptide to circumvent the need forantibody detection. Antibody detection can present disadvantages due tovariability in detection depending on epitope availability of the Aβoligomer when it is bound to transfer membranes. We anticipate that useof biotinylated peptide will provide us with greater sensitivity andreproducibility since antibody-epitope recognition is not required;rather very high affinity biotin-streptavidin interactions are utilizedfor detection.

Aβ(1-40) peptide was obtained with biotin coupled to its N-terminus,since structural determinations suggest that the C-terminal residuesrepresent a primary site for β-sheet transitions and oligomerization(Soto, 1999, Mol Med Today 5:343-350; Jarrett et al., 1993, Ann NY AcadSci 695:144-148; Jarrett et al., 1993, Biochemistry 32:4693-4697). Theexpectation that biotin attached to the N-terminus of the peptide wouldhave little effect on the oligomerization process was confirmed asfollows: Biotin-Aβ(1-40) peptide was diluted to a final concentration of20 μg/ml into phosphate buffered saline, pH 6.0 and incubated for 48 hat 37° C. in the presence or absence of selected curcumin analogs.Following incubation, the peptide solutions were centrifuged (12,000×g,2 min) and mixed with an equal volume of 2-fold concentrated Tricinesample buffer and fractionated by electrophoresis on 10-20% precastTris-Tricine polyacrylamide gels. Fractionated material was thentransferred to PVDF membranes using a semi-dry transfer unit (20V, 40min) and non-specific sites were blocked by incubation with 20 mM Tris,pH 7.4, 150 mM NaCl, 0.1% Tween-20 (TBS-T), 5% calf serum. Membraneswere then incubated streptavidin conjugated with horseradish peroxidaseand processed for chemiluminescence detection. Images were capturedusing a Syngene GeneGnome system equipped with a Peltier-cooled 16-bitCCD camera and saturation detection. Without curcumin treatment, wefound high molecular weight aggregates resolved by Tris-Tricine PAGEanalysis (FIG. 35).

These oligomers ranged in relative molecular mass from 15-75 kDa, as wasseen by Yang, et al. (Yang et al., 2005, J Biol Chem 280:5892-5901).When Aβ peptide was incubated with varying concentrations of curcumin,we found that 2.0 and 20 μM concentrations completely inhibited Aβoligomer formation, whereas 0.5 μM had little effect. Usingnon-biotinylated Aβ(1-40), Yang, et al., reported an IC₅₀ value forcurcumin of 1 μM, which is similar to our estimation obtained from PAGEanalysis. These results clearly demonstrate that curcumin preventsoligomerization of N-terminal biotinylated Aβ(1-40) peptide in anidentical manner as that shown by Yang, et al. (Yang et al., 2005, JBiol Chem 280:5892-5901) using non-biotinylated peptide.

We next examined the effect of 4 structurally different analogs ofcurcumin to determine if oligomer inhibition was restricted to curcuminor if variations could be introduced into its molecular structure thatmight assist us in identifying the essential chemical groups responsiblefor its inhibitory properties and guide us in the design of moreefficacious inhibitors. From our library of 84 analogs, we chose analogs(Ana) 1-4 for our initial limited screening. These were selected basedon their substitutions and degree of unsaturation of the carbon spacer(FIG. 36). As shown in FIG. 36, Ana 1 contains two aryl rings and asaturated 7-carbon spacer designed to test the importance ofunsaturation in the spacer arm; Ana 2 contains two aryl rings and anunsaturated 3-carbon spacer to test the importance of spacer length; Ana3 contains two aryl rings separated by an unsaturated 7-carbon spacerand an additional aryl ring attached to the central methylene carbon totest if additional aryl ring structures impact inhibitory effectiveness;and Ana 4 contains two aryl rings separated by a fully saturated7-carbon spacer and an aryl ring attached to the central methylenecarbon designed to test the importance of unsaturation in the spacer armtogether with an additional aryl ring structure.

We applied the same experimental protocol using the 4 different analogsas that described in FIG. 36 for curcumin. Importantly, we found thatall 4 analogs demonstrated some degree of inhibition of Aβoligomerization (FIG. 36). Ana 1 and Ana 4 both inhibitedoligomerization with an IC₅₀<20 μM, indicating the α,β-unsaturatedcarbon spacer is not essential to maintain inhibitory properties. Thisis an important observation due to the potential of α,β-unsaturatedgroups serving as Michael acceptors. The original work on curcumin byYang, et al. (Yang et al., 2005, J Biol Chem 280:5892-5901) failed toaddress this critical concern. Ana 2 also inhibited Aβ oligomerizationwith an IC₅₀ of 20 μM. Although not as effective as curcumin, Ana 2 onlyhas a 3-carbon spacer between the phenolic rings as opposed to the7-carbon spacer of curcumin suggesting that the bi-phenolic structuremay provide the greatest contribution toward the inhibitory properties.These data also demonstrate flexibility in ring spacing since spacerdistance can be varied between 3 and 7 carbons. Finally, Ana 3demonstrates an IC₅₀ value <2 μM and is at least as effective ascurcumin in preventing Aβ oligomerization. Ana 3 closely resemblescurcumin with the addition of an aryl ring attached at the centralmethylene carbon. The addition of this hydrophobic ring clearly does notinterfere with the inhibitory properties of the basic curcumin structureand once accurately titrated in an ELISA-based assay, may prove moreeffective than curcumin.

From these preliminary results, we are beginning to obtain a picture ofthe necessary features in the molecular structure of curcumin that areresponsible for its inhibitory properties toward Aβ oligomerization. Itis our goal to develop more effective aggregation inhibitors bycapitalizing on the newly established inhibitory properties of curcumin.Curcumin itself demonstrates IC₅₀ values in the low micromolar rangeleaving much room for improvement. Once we identify the criticalfeatures of curcumin that are responsible for prevention ofoligomerization, we believe developing synthetic inhibitors withnanomolar IC₅₀ values are very possible given the success of ourpreliminary studies. Developing inhibitors with nanomolar IC₅₀ values isimperative when taking into consideration some basic facts of thephysiological properties of curcumin. First, curcumin is poorly absorbedby the intestinal epithelium (Ammon et al., 1991, Planta Med 57:1-7;Ravindranath et al., 1980, Toxicology 16:259-265; Sharma et al., 2001,Clin Cancer Res 7:1894-1900) and this is probably the reason whyingesting large doses of curcumin are well tolerated in clinical trials(Chainani-Wu, 2003, J Altern Complement Med 9:161-168). Second, closerexamination of the effects of dietary curcumin on in vivo amyloid plaqueburden presented by Yang, et al. (Yang et al., 2005, J Biol Chem280:5892-5901), reveals that, although curcumin significantly reducedhippocampal plaque burden, this effect was limited to a modest 32.5%reduction. We believe that the percent reduction in plaque burden can beincreased with analogs demonstrating greater IC₅₀ values to offset thepoor uptake by intestinal epithelia. Modifications we make to the basecurcumin structure may also contribute to the dietary uptake, which willonly serve to enhance its biologic effects on Aβ oligomer dissolution.

In order to identify the molecular features of curcumin that areresponsible for inhibition of Aβ peptide oligomerization using chemicalanalogs of curcumin, we will determine whether our library ofcurcumin-based analogs contains compounds that can inhibit Aβ peptideaggregation and disaggregate preformed Aβ fibrils at sub-micromolarconcentrations. Curcumin is a symmetrical diphenolic dienone that existsin equilibrium between diketo and keto-enol forms. We have synthesized aseries of three enone analogs of curcumin totaling over 80 compoundsthat include those: 1) that retain the 7-carbon between the aryl rings,2) with a 5-carbon spacer, and 3) with a 3-carbon spacer (Weber et al.,2005, Bioorg Med Chem 13:3811-3820). We will compare each of thesecompounds with curcumin in their abilities to inhibit Aβ peptideaggregation and to disaggregate preformed Aβ oligomers using two invitro assays. The first assay described below is a novel ELISA-basedscreening procedure that will allow us to accurately titrate theeffectiveness of each curcumin-analog toward preventing Aβoligomerization. The antibody we will employ for this assay specificallyrecognizes the oligomeric conformation of the Aβ aggregate, but not thefibrillar or monomeric forms (Glabe, 2004, Trends Biochem Sci29:542-547; Kayed et al., 2003, Science 300:486-489). We plan tocontinue using the biotinylated Aβ peptide and use streptavidin-coatedplates for capture, rather than anti-Aβ antibodies to ensure efficientbinding of aggregates that might otherwise be precluded due to sterichindrance caused by the oligomeric structure.

Other in vitro assays such as use of thioflavin, which rely upon afluorescent shift following aggregate binding (LeVine 3rd, 1999, MethodsEnzymol 309:274-284; Naiki et al., 1989, Anal Biochem 177:244-249), arenot practical for our needs because excitation/emission spectra forthioflavin are similar to those of our curcumin-based compounds. Usingour ELISA-based assay, which will provide us with a rapid, reproducibleand cost-effective screening protocol, we fully anticipate identifyingcompounds with equal or greater potential than curcumin toward Aβaggregate disruption. Once identified, we will test these compounds fortheir ability to prevent Aβ oligomerization using SDS-PAGE analysis.

Although we plan to concentrate our initial efforts screening theanalogs in our current library, we are not limited to the 84 compoundspresently on hand. The syntheses of these 3-, 5-, and 7-carbon spaceranalogs, as well as incorporating varying degrees of saturation, arestraight forward and the schemes are adaptable. The availability of alarge number of substituted benzaldehydes, heterocyclic ring-containingaldehydes and substituted acetophenones adds efficiency and flexibilityto the synthetic procedures. In addition, many of the syntheticreactions require only one or two steps as illustrated in FIG. 37.Series I analogues, maintaining the 7-carbon dienone spacer between thearomatic rings as in curcumin, were synthesized from aromatic andhetercyclic aldehydes by condensation with 2,4-pentanedione in an aldoltype reaction (Pabon, 1964, Recueil 83:379-386). This involvesbase-catalyzed condensation in the presence of a trialkylborate tocomplex with the carbonyl groups, which prevents enolization and guidesthe reaction. If a mixture of two different aldehydes is used,unsymmetrical analogues are formed. Series II analogues, containing a5-carbon enone spacer, were synthesized either by base-catalyzedcondensation of the appropriate aldehyde with acetone or byacid-catalyzed condensation with 3-oxo-glutaric acid (Masuda et al.,1993, Phytochemistry 32:1557-1560; Zelle et al., 1998, World Patent,9820891). Series III, containing a 3-carbon enone spacer weresynthesized by base-catalyzed condensation of the appropriate aldehydewith a substituted acetophenone (Kohler et al., 1932, Org. Synth., Coll.Vol. 1).

Experimental design: N-terminal biotinylated-Aβ(1-40) peptide will bedissolved in dimethylsulfoxide (DMSO) to a final concentration of 5mg/ml and sonicated for 30 min. This material will be then centrifugedthrough a 0.2 μm spin-filter and stored at −80° C. Sonication andfiltration are necessary to remove any trace of undissolved seeds thatmay nucleate aggregate formation (Evans et al., 1995, Proc Natl Acad SciUSA 92:763-767). This preparation remains stable and aggregate-free at−80° C. Immediately before use, the peptide will be diluted to a finalconcentration of 20 μg/ml into phosphate buffered saline (PBS), pH 6.0and incubated for 48 h at 37° C. in the presence or absence of varyingconcentrations of curcumin or curcumin-based analogs (0.01-20 μM).Following this incubation, the reactions will be centrifuged (12,000×g,5 min) and added to wells of streptavidin-coated 96-well plates for 1 hat 23° C. Wells will be rinsed 3 times with PBS, pH 7.2, to removeunbound biotinylated material and blocked to prevent non-specificadsorption with PBS containing 0.1% Tween-20, 5% calf serum (blockingbuffer) for 1 h, 23° C. Wells will then be incubated with rabbitpolyclonal antibody A11 (1 μg/ml, available from BioSourceInternational, Camarillo, Calif.) for 1 h, 23° C., followed by rinsingwith PBS containing 0.1% Tween-20 and incubated with HRP-conjugated,goat anti-rabbit secondary antibody for 1 h, 23° C. Unbound secondaryantibody will be removed by rinsing wells with PBS, 0.1% Tween-20 anddeveloped with peroxidase substrate, 3,3′,5,5′-tetramethylbenzidine(TMB). The reaction will be terminated by the addition of 1 N sulfuricacid and spectrophotometric readings will be taken at 450 nm forquantitation. Positive controls will include peptide incubated in theabsence of inhibitors to permit fibril formation, and negative controlswill consist of peptide freshly diluted just prior to adding reactionmixtures to streptavidin-coated plates. All assays will be performedusing varying dilutions of each compound in order to determine IC₅₀values for accurate comparison with native curcumin.

We will then determine whether curcumin-based analogs identified asinhibitors of Aβ peptide aggregation are also able to inhibit Aβoligomerization. Since soluble Aβ oligomers are more diffusible thanamyloid fibrils and viewed as playing an important role in ADpathogenesis, we will also examine if our curcumin analogs are capableof preventing the formation of peptide oligomers. Visualizing Aβoligomers is best accomplished by Tris-Tricine based SDS-PAGE analysis(Yang et al., 2005, J Biol Chem 280:5892-5901). Using this method, Aβmonomers (˜4 kDa) are readily distinguished from 4-5-mers, as well ashigher molecular weight oligomers that range in relative molecularmasses from 44-127 kDa (Yang et al., 2005, J Biol Chem 280:5892-5901).To permit better comparisons with results obtained by ELISA assays, andfor sensitivity in detection, we will continue to use our biotinylatedAβ(1-40) peptide. Toward this end, peptide will be incubated in thepresence or absence of varying concentrations of curcumin analogs and,following incubation, resolved by Tris-Tricine SDS-PAGE. Peptide andoligomers will then be transferred to PVDF membrane and probed withHRP-conjugated streptavidin, followed by chemiluminescence detection. Weanticipate that one or more of the curcumin analogs identified in ourELISA capture assay will also inhibit Aβ peptide oligomerization in aconcentration-dependent manner. With densitometric analyses, we willcalculate IC₅₀ values for oligomer inhibition and compare these valuesdirectly to fibril inhibition derived from our ELISA-based assay.

Experimental design: Biotinylated-Aβ(1-40) peptide will be diluted to afinal concentration of 20 μg/ml into PBS, pH 6.0 and incubated for 48 hat 37° C. in the presence or absence of varying concentrations ofcurcumin or curcumin-based analogs (0.01-20 μM). Following incubation,the reactions will be mixed with an equal volume of 2-fold concentratedTricine sample buffer without reducing agents and separated on 10-20%Tris-Tricine SDS gels. Peptides will then be transferred to PVDFmembranes and incubated with PBS containing 0.1% Tween-20, 5% calf serumfor 1 h, 23° C. Membranes will then be incubated with HRP-conjugatedstreptavidin and processed for chemiluminescence detection according tomanufacturer's instructions (SuperSignal, Pierce). Images will becaptured using a Syngene GeneGnome system equipped with a Peltier-cooled16-bit CCD camera. This camera has full saturation detection and a largedynamic linear range for accurate quantitation of chemluminescentsignals. Densitometric analysis will be performed using Scion Image,version 4.0.2. All experiments will be done in triplicate and data willbe plotted and subjected to regression analysis to determine IC50values.

We will then determine whether the curcumin-based analogs directly bindAβ peptide aggregates and, if so, with what affinity. Curcumin has beenshown to bind directly to Aβ fibrils and demonstrate little or noaffinity for Aβ monomers (80). However, in that study the experimentaldesign was such that affinity measurements could not be made.Determining binding affinity for curcumin and chemical analogs iscritical to evaluating their mechanism of inhibition and for futureimprovements upon inhibitor design. Therefore, we will test curcumin andidentified analogs that inhibit Aβ peptide aggregation for theirabilities to bind Aβ fibrils and determine affinity measurements fordirect comparisons. Toward this end, we will incubatebiotinylated-Aβ(1-40) peptide to form aggregates and after which capturethe aggregates on streptavidin-coated 96-well plates. Curcumin andcurcumin analogs will then be incubated with immobilized aggregates andbinding will be quantitated by fluorescence detection taking advantageof the fluorescent properties of curcumin and its chemical analogs.Quantitation of direct binding will allow us to calculate the affinityconstant for each inhibitor which will first confirm if the analogs areable to bind Aβ aggregates directly and second, determine if theinteraction is of high- or low-affinity.

Experimental design: Biotinylated-Aβ(1-40) peptide will be diluted to afinal concentration of 20 μg/ml into PBS, pH 6.0 and incubated for 48 hat 37° C. to permit aggregation. Reaction mixtures will then beincubated in 96-well plates pre-coated with streptavidin for 2 h at 23°C. to capture biotinylated aggregates. Wells will be rinsed 3 times withPBS, pH 7.2 to remove unbound material, followed by incubation withvarying concentrations of curcumin or curcumin analogs for 2 h at 23° C.A two hour-incubation period is chosen since this time is insufficientfor disaggregation of preformed fibrils, but sufficient to permitcurcumin binding to aggregates (Yang et al., 2005, J Biol Chem280:5892-5901). Unbound curcumin or curcumin analogs will be removed byrinsing the membrane 3 times with PBS and bound material will bequantitated by fluorescence detection using a fluorescence plate reader(excitation/emission, 355/518 nm). All points will be carried out intriplicate and data will be subjected to regression analysis todetermine binding affinities.

We will next determine whether curcumin or its chemical analogsdestabilize the β-sheet conformation of Aβ peptide aggregates andstabilize the non-aggregated α-helical/random coil conformation. Becauseof the chiral properties of the Aβ peptide backbone, circular dichroismspectroscopy can be used to determine its absorption spectra in the farUV range and obtain information about the content of α-helix, β-sheet orrandom coil within the peptide structure. The wavelengths used rangefrom 190-250 nm, which are well below the excitation/emission spectra ofcurcumin in an aqueous environment (λex=355 nm, λem=518 nm) (Khopde etal., 2000, Photochem Photobiol 72:625-631) or its analogs, thus thepresence of these compounds in the sample is not expected to interferewith the measurements. These measurements will provide criticalinformation that will enable us to better define the mechanism ofcurcumin action. How curcumin disaggregates the Aβ fibril structure iscurrently unknown. Since Aβ aggregation requires a conformational shiftfrom α-helix/random coil to β-sheet to seed the aggregation process, andas curcumin is known to directly bind Aβ aggregates, we hypothesize thatcurcumin may bind to and de-stabilize β-sheet conformers, therebyshifting the equilibrium to a greater α-helical/random coil population.To test this hypothesis, we plan to take circular dichroism measurementsof Aβ peptide in the presence of varying concentrations of curcumin orits chemical analogs. In the absence of inhibitor, we anticipatemeasuring predominantly β-sheet conformation of the fibril solution asprevious studies have shown (Roher et al., 2000, Biochim Biophys Acta1502:31-43, Bieler et al., 2004, Curr Drug Targets 5:553-558; Lopez DeLa Paz et al., 2002, Proc Natl Acad Sci USA 99:16052-16057; Xu et al.,2005, Proc Natl Acad Sci USA 102:5403-5407). With increasing inhibitorconcentration, we expect to observe a shift toward α-helical/random coilcontent, the amount of which will be dependent upon inhibitor structureand concentration.

To ensure that the curcumin- and analog-dependent conformational changeswe measure by CD analysis are accompanied by a loss of fibril andoligomeric structure, we will also examine aliquots of Aβ peptideincubated with or without aggregation inhibitors by electron microscopy.We anticipate that a loss of fibril structure resulting from incubationwith the aggregation inhibitors will closely correlate with aconformational shift from β-sheet to α-helix/random coil. Suchultrastructural studies will not only confirm the disaggregationproperties of our newly discovered inhibitors, but also provideadditional mechanistic evidence correlating loss of β-sheet structurewith loss of fibril structures.

Experimental design: Biotinylated-Aβ(1-40) peptide will be diluted to afinal concentration of 20 μg/ml into PBS, pH 6.0 and incubated for 48 hat 37° C. in the presence or absence of varying concentrations ofcurcumin or curcumin-based analogs. A minimum of 20 CD scans will beacquired in the range of 190-250 nm by taking points every 0.2 nm. Ascan rate of 100 nm/min with band width of 1 nm will be used.

Electron microscopy analysis: A sample of the Aβ aggregates incubated inthe presence or absence of curcumin or its analogs will be adsorbed toformvar-coated 400-mesh copper EM grids for 30 min. The grids will thenbe stained with 2% uranyl acetate and examined with a Hitachi 600electron microscope.

In order to determine whether analogs of curcumin are more effectivethan curcumin in reducing cytotoxicity caused by Aβ oligomers incultured neuronal cells, we will first determine whether thecurcumin-based analogs protect against Aβ aggregate-mediatedcytotoxicity. AD is ultimately a neurodegenerative disease as it islargely accepted that the progressive deposition of Aβ is directly toxicto neurons and increases their susceptibility to oxidative and metabolicstress, and excitotoxicity (Mattson, 1997, Physiol Rev 77:1081-1132).These insults significantly impact synaptic plasticity and markedlyinhibit long-term potentiation. Because of this, it is imperative thatinhibitors of Aβ fibrillogenesis be tested for their ability to protectneuronal cells from aggregate-induced cytotoxicity if they are to beconsidered of any potential therapeutic value. Therefore, we willdetermine if the curcumin analogs we identify as Aβ aggregate inhibitorsare neuroprotective. Toward this end, we will carry out twowell-characterized assays to assess: 1) protection against Aβoligomer-induced cell death by measuring lactate dehydrogenase (LDH)release, and 2) analog-dependent increase in cell viability bydetermining metabolic activity using the MTT[3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] reductionassay. LDH release is correlated to disruption of cellular integrity anda direct measure of cell death. MTT, a tetrazolium salt, undergoesreduction by dehydrogenases thereby generating intracellular formazanwhich can be measured spectroscopically. For these studies, we will usethe neuroblastoma-derived cell line, SH-SY5Y, which have been previouslyused for aggregation-dependent cytotoxicity measurements (Datki et al.,2003, Brain Res Bull 62:223-229). Upon addition of retinoic acid (RA) tothe culture media, these cells undergo morphological differentiation,growth arrest and develop characteristics of a distinct neuronalphenotype (Pahlman et al., 1984, Cell Differ 14:135-144).

Experimental design: SH-SY5Y cells will be induced to differentiate bysupplementing media with 10 μM all-trans-retinoic acid for 7 days.Following differentiation, cells will be incubated with preformed Aβoligomers (100 nM) in the presence or absence of varying concentrationsof curcumin (as our positive control) or curcumin analogs for 48 h at37° C. LDH assays will then be performed on culture media and MTT assayswill be performed on cells.

LDH assay: Standard, reliable kits for measuring LDH activity arecommercially available (Promega, Sigma-Aldrich and CalBiochem).

MTT assay: 500 μg/ml MTT is prepared in RPMI-1640 media without phenolred and added to cells for 2-4 h at 37° C. until purple precipitate isseen. Media is then removed and dye is extracted with acidic isopropanol(0.04 M HCl in absolute isopropanol), and absorbance is taken at 570 nmwith background subtraction at 650 nm. All experimental points will bedone in triplicate to establish statistical significance.

We will then determine whether the curcumin-based analogs protectagainst Aβ aggregate-mediated glial cell activation. Glial cellactivation, both astrocytes and microglia, with subsequent production ofproinflammatory mediators, is thought to play a significant role in thepathophysiology of AD (McGeer et al., 1995, Brain Res Brain Res Rev21:195-218). A number of inflammatory effectors are secreted bymicroglia in AD brain including nitric oxide, interleukin-6 (IL-6), andtumor necrosis factor-α, among others (Lue et al., 2001, Glia 35:72-79).The centers of these inflammatory responses are the amyloid deposits(Selkoe, 1991, Neuron 6:487-498; Mehlhorn et al., 2000, Int J DevNeurosci 18:423-431). Importantly for our objectives, Aβ aggregates havebeen shown to induce the production of inflammatory mediators incultured glial cells (Araujo et al., 1992, Brain Res 569:141-145;Goodwin et al., 1995, Brain Res 692:207-214; Meda et al., 1995, Nature374:647-650). Rat C6 glioma cells are a useful model for Aβaggregate-induced cytokine production (Bales et al., 1998, Brain Res MolBrain Res 57:63-72; Hu et al., 1998, Brain Res 785:195-206; Pena et al.,1995, Brain Res Mol Brain Res 34:118-126) and with these cells we willexamine if curcumin analogs, identified as inhibitory for peptideaggregation, protect against glial cell activation. We will quantitatesecreted levels of IL-6 from cultured C6 cells as a measure ofinflammatory activation. We anticipate that Aβ-aggregates will induceIL-6 production in these cells, as reported previously (Hu et al., 1998,Brain Res 785:195-206; Pena et al., 1995, Brain Res Mol Brain Res34:118-126; Gasic-Milenkovic et al., 2003, Eur J Neurosci 17:813-821),and our curcumin analogs will protect cells from activation by virtue ofaggregate dissolution. These data will provide important informationregarding additional functions for curcumin analogs; in addition to Aβpeptide aggregate dissolution, the analogs may also provide aneuroprotective function by preventing induction of harmful inflammatorymediators.

Experimental design: Rat C6 glioma cells will be seeded in 96-wellplates at a density of 5×10⁴ cells per well and grown for 24 h. Culturemedia will be replaced with 25 μM Aβ peptide with or without varyingconcentrations of curcumin or curcumin analogs for 48 h. Culturesupernatants will be tested for IL-6 secretion using an ELISA specificfor rat IL-6 (available from BioSource, Inc.). This particular ELISAsystem is sensitive to <8 μg/ml IL-6 with a working range of 30-2000pg/ml.

Next, we will determine whether the curcumin-based analogs that preventAβ aggregation and glial cell activation exhibit lipid membranepermeability. Successful drug development methodologies require closeattention to physicochemical properties that ultimately dictatebioavailability. Knowledge obtained regarding these properties, whichinclude absorption, distribution, metabolism and excretion (ADME), arecritical for effective and useful drug development. We will carry out apre-ADME screening process on curcumin analogs that we identify aseffective in inhibiting Aβ aggregation and protective in neurotoxicityassays. A pre-ADME screening system is commercially available thatpermits early characterization of lead compounds in the drug developmentprocess. Since lipophilicity is a major determinant of a compounds'sinherent bioavailability, the assay quantitatively determines lipidpermeability using artificial membranes prepared with a mixture ofphospholipids designed to mimic intestinal brush-border membranes (Kansyet al., 1998, J Med Chem 41:1007-1010; Sugano et al., 2001, J BiomolScreen 6:189-196). In addition, passive drug diffusion results will beobtained using artificial membranes of hexane/hexadecane (Wohnsland etal., 2001, J Med Chem 44:923-930). Since our analogs are based onmulti-aryl ring structure, we anticipate that each one will display ameasurable permeability in these assays. We will compare these resultsdirectly to those obtained with curcumin, which will provide us with acomparative assessment with a compound that has already demonstratedsome degree of bioavailability and passage of the blood-brain barrier.

Experimental Design:

Parallel Artificial Membrane Permeation Assay (PAMPA): Each well of a96-well “donor” plate will receive 5 μl lipids prepared in organicsolvent (e.g. 2% lecithin in dodecane). Lipids will be of a desiredcomposition to mimic intestinal brush-border membranes. PBS containing5% DMSO at the desired pH will be added to the “acceptor” plate. Analogsof interest (150 μl) will be prepared at varying concentrations in 5%DMSO/PBS and added to the “donor” plate. The pH of this solution will bevaried to resemble intestinal pH changes that occur throughout itslength in order to assess the effect of pH on absorption. The “donor”plate is then nested into the “acceptor” plate ensuring that theunderside of the lipid membrane is in contact with buffer. The plateswill be incubated at 23° C. for 16-24 hours. Absorption will be measuredon 100 μL/well from the “donor” plate and 250 μL/well from the“acceptor” plate. Integrity of the artificial lipid membrane will bemeasured by quantitating the permeability of Lucifer Yellow. This dyedemonstrates poor lipid permeability and should be incapable ofdiffusion into the “acceptor” plate. Lucifer Yellow will be quantitatedby fluorescence analysis (λex 425 nm, λem 528 nm).

Passive Drug Permeability Assay: Each well of a 96-well “donor” platewill receive 15 μl of a 5% solution (v/v) of hexadecane in hexane.Plates are then allowed to dry for 1 h in a fume hood to ensure completeevaporation of the hexane allowing for the formation of a uniform layerof hexadecane. PBS containing 5% DMSO is added to each well of the“acceptor plate” and the hexadecane-treated “donor” plate is placed intothe acceptor plate ensuring that the underside of the membrane is incontact with the buffer. Analogs of interest will be prepared at varyingconcentrations in 5% DMSO/PBS and added to the “donor” plate andincubated at 23° C. for 5 hours. Absorption is then measure for samplesfrom both “donor” and “acceptor” plates. Finally, we will continue toimprove upon the efficacy of chemical analogs of curcumin usingligand-based drug design.

Example 11 Endothelial Targets and Prevention of Diabetic Complications

Summary

Inflammation and oxidative stress play a major role in the endothelialdysfunction that is associated with diabetes and its microvascularcomplications. The transcription factor nuclear factor κB (NF-κB) iswell known as a regulator of genes controlling the inflammatoryresponse. NF-κB is commonly activated in endothelial cells in diabetes.We propose that limiting the activation of NFκB may be a new approach tothe development of therapies for prevention of the microvascularcomplications of diabetes. We will develop compounds related tocurcumin. The polyphenol curcumin, which is the active compound in thespice turmeric, has been used for centuries in Asia as a medicinal fortreatment of a wide variety of health problems including inflammation.More recently, curcumin has been found to inhibit activation of NF-κB.

We intend to demonstrate that NF-κB is activated in endothelial cells inresponse to exposure to agents associated with microvascularcomplications and that activation of NF-κB results in up-regulation ofgenes associated with endothelial dysfunction. Human retinal endothelialcells will be exposed to agents or conditions that promote endothelialdysfunction, such as: high glucose; Advanced Glycation Endproducts;angiotensin II; and TNF-α. Activation of NF-κB and up-regulation ofgenes associated with the pro-inflammatory state (IL-1, IL-6), withextracellular matrix production (collagen, fibronectin) and withleukocyte adherence (E-selectin, ICAM-1) will be determined. Geneticmanipulation of the NF-κB pathways will be used to determine ifactivation of NF-κB is required for the increased expression of thesegenes that promote endothelial dysfunction.

We will also evaluate curcumin and its analogs as potential therapeuticsto prevent activation of NF-κB and to demonstrate that these inhibitorsprevent development of endothelial dysfunction. We have developedanalog-libraries of curcumin analogs and have demonstrated that a numberof these analogs are more active than the parent compounds as inhibitorsof the activation of NF-κB. Moreover, we have demonstrated that theanti-oxidant activity of these analogs is not required for inhibition ofNF-κB. Endothelial cells will be used to determine the ability of theseanalogs to inhibit the activation of NF-κB and to prevent theNF-κB-dependent up-regulation of genes that promote endothelialdysfunction.

Endothelial Dysfunction and Diabetic Complications

Endothelial cells (EC), by virtue of their location between thecirculation and the vascular tissue, are able to detect humoral changesand to transmit this information to other vascular cells throughmechanisms involving altered gene expression both in the EC and in thetarget cells. EC responses to humoral changes include altering theexpression of growth factors, cytokines and adhesion molecules. Thus theendothelium actively regulates vascular tone and permeability, balancescoagulation and fibrinolysis, controls the composition of subendothelialmatrix, regulates adhesion and extravasation of leukocytes and therebyinfluences inflammatory activity in vessel walls. Considerable evidencelinks EC dysfunction with development of microvascular complications intype 1 and type 2 diabetes (Yamagishi et al., 2005, Curr Pharm Des11:2279-2299; Schalkwijk et al., 2005, Clin Sci 109:143-159; Pomilio etal., 2002, J Pediatr Endocrinol Metab 15:343-361; Hink et al., 2003,Treat Endocrinol 2:293-304). EC dysfunction in diabetes is linked tohyperglycemia (Stenina, 2005, Curr Pharm Des 11:2277-2278). Glucosetransport into EC is insulin-independent and appears not to beregulated. Consequently, EC experience high internal concentrations ofglucose in response to hyperglycemia with the resulting accumulation ofvarious glucose metabolites. When exposed to high glucose in vitro, ECup-regulate the production of matrix components, including collagen andfibronectin (Cagliero et al., 1988, J Clin Invest 82:735-738), and ofpro-coagulant proteins (Boeri et al., 1989, Diabetes 38:212-218). ECexhibit a decrease in proliferation, fibrinolyic potential and increasedapoptosis in response to high glucose (Graier et al., 1995, Eur JPharmacol 294:221-229; Maiello et al., 1992, Diabetes 41:1009-1015), andthey up-regulate expression of TGF-β1 which likely controls theexpression of many of the genes that code for extracellular matrix(Yevdokimova et al., 2004, J Diabetes Complications 18:300-308). EC alsorespond to changes in circulating cytokines. The cytokine VEGF, forexample, can stimulate differentiation, survival, migration,proliferation and permeability in EC and is especially associated withdiabetic retinopathy (Benjamin, 2001, Am J Pathol 158:1181-1184).Likewise, diabetic nephropathy is associated with expression ofinflammation markers such as CRP, fibrinogen and IL-6, and withincreased expression of adhesion molecules such as ICAM-1, which promoteinflammation by increasing leukocyte adherence and infiltration (DallaVestra et al., 2005, J Am Soc Nephrol 16:S78-S82). The response of EC tothese cytokines commonly involves signaling through transcription factorNF-κB. Additionally, oxidative stress has consistently been shown inexperimental models of diabetes (Mohamed et al., 1999, BioFactors10:157-167), and activation of NF-κB is often observed in response tothese stresses.

Curcumin: A Natural Inhibitor of the Activation of NFκB

For centuries, curcumin has been used in India and Southeast Asia as amedicinal for a wide variety of conditions. Curcumin has been reportedto possess antioxidant, anti-inflammatory, antiviral, andantimutagenesis activities (Araujo et al., 2001, Mem Inst Oswaldo Cruz96:723-728). A number of recent studies of curcumin in experimentalmodels of diabetes have reported beneficial effects of these naturalproducts, which were assumed to be due to their anti-oxidant properties(Suranarayana et al., 2005, Invest Ophthalmol Vis Sci 46:2092-2099; Arunet al., 2002, Plant Foods Hum Ntr 57:41-52). However, curcumin canprevent the stress-induced activation of NF-κB in a variety of cells(Bremner et al., 2002, J Pharm Pharmacol 54:453-472; Aggarwal et al.,2004, Ann NY Acad Sci 1030:434-441; Shimizu et al., 2005, Mutat Res591:147-160), which has been suggested to be the result of inhibition ofIKK or of a kinase that activates IKK (Bharti et al., 2003, Blood101:1053-1062). Since curcumin is a potent antioxidant, the multiplebiological activities of these polyphenolic natural products may reflecttheir general anti-oxidant properties and/or these compounds may betargeted to specific proteins, such as kinases that regulate theexpression of NF-κB. This question is especially relevant to the issueof the role of NF-κB in EC dysfunction in response to hyperglycemiawhere, as discussed above, oxidative stress often appears inseparablefrom activation of NF-κB. To address this question, initial studies werecarried out to design libraries of analogs of curcumin, to evaluate theanti-oxidant properties of these analogs, and to test whether theability of these analogs to prevent activation of NF-κB requiredretention of their anti-oxidant properties.

Preliminary Studies

The observation that simple compounds such as curcumin can blockactivation of NFκB leads to the hypothesis that synthetic compounds withenhanced activity compared to curcumin can be developed that block NFκBactivation. These compounds would be anticipated to exhibitanti-inflammatory activity in EC exposed to high glucose and relatedstresses associated with diabetic complications and to prevent theup-regulation of genes that promote EC dysfunction. As mentioned above,the biological activities of curcumin are broad and have often beenassociated with their anti-oxidant activities. On the other hand, thesuggestion that these natural products prevent the activation of NF-κBby inhibiting IKK or upstream kinases implies that there are specifictargets of curcumin. The anti-oxidant activities of curcumin are derivedfrom the phenolic functional groups. Therefore, synthesis of analogsdevoid of the phenolic groups should alter their anti-oxidant activitesand should provide analogs that can be used to test whether anti-oxidantactivity is necessary for inhibition of the activation of NF-κB. Thepreliminary results below (recently published, Weber et al., 2005,Bioorg Med Chem 13:3811-3820; Weber et al., 2006, Biorg Med Chem14:2450-2461) were designed to 1) develop efficient reaction schemes forthe synthesis of analogs of curcumin; 2) evaluate the anti-oxidantproperties of these analogs; 3) compare these analogs for theirabilities to inhibit the activation of NF-κB; and 4) determine whetheranti-oxidant activity is required for inhibition of NF-κB. These studieswere conducted using a commercial NF-κB reporter stable cell linedesigned for screening inhibitors of NF-κB.

Synthesis and Biological Activity

Curcumin analog library: We have constructed a chemical library ofcurcumin analogs; these were used to identify specific functional groupsresponsible for curcumin's established anti-oxidant properties. Theseanalogs, in general, retain the enone functionality of curcumin. Theanalogs include: 1) those that retain the 7-carbon spacer between thearyl rings as in curcumin; 2) those with a 5-carbon spacer; and 3) thosewith a 3-carbon spacer. In addition to carbon spacer variations, analogswere synthesized that have enone or dienone functionality, varyingdegrees of unsaturation in the spacer, and addition of alkyl or arylgroups to the spacer. We also have synthesized analogs with limitedrotational flexibility and with heterocyclic aromatic rings. A briefsummary of the synthetic schemes is presented in FIG. 37. These reactionschemes are versatile and efficient, providing us the ability to developpreliminary Structure-Activity Relationships (SAR) and, based uponinitial screening with the Panomics NF-κB Reporter Stable Cell Line, theability to utilize the screening data to design new analogs forhypothesis testing. The chemical basis of the anti-oxidant activities ofcurcumin and analogs, along with the synthetic schemes, have beenreported (Weber et al., 2005, Bioorg Med Chem 13:3811-3820).

Inhibition of the Activation of NF-κB by Analogs of Curcumin

We carried out a screen of our curcumin library, using the commercialPanomics NF-κB Reporter Stable Cell; this is the human 293T embryonickidney cell line stably transfected with the luciferase gene controlledby an NFκB-dependent promoter (293T/NFκB-luc). This cell line wasdeveloped by Panomics for screening potential inhibitors of NF-κB. Cellsare stimulated with TNF-α to express luciferase, whose activity ismonitored in a chemiluminometer. Screening of curcumin and analogsinvolves determining the ability of the analogs to inhibit theactivation of NFκB, which is detected as diminished chemiluminescence.All of the analogs were separately analyzed for their effects on cellviability and growth and were demonstrated to be non-toxic at theconcentrations used in screening. In this preliminary study, weidentified a number of analogs that are more active than curcumin (Table12).

Several points are noteworthy: 1) Derivatives of curcumin, such asanalog 2 that contains an alkyl group attached to the central carbon,can retain activity; a large number of different alkyl or aralkyl groupscan be attached at this position; 2) Analogs with activity can readilybe identified in the C7 (1 and 2) and C5 (3, 4, and 5) series ofanalogs; 3) Even analogs with heterocyclic rings can retain activity(analog 3); and 4) Analogs that do not exhibit anti-oxidant activity,such as 3 and 5, can prevent activation of NFκB. This demonstrates thatthe biological activities of curcumin analogs can be separated fromtheir anti-oxidant activities. This suggests that these analogs havespecific biological targets rather than acting as general anti-oxidants.

TABLE 12 Compound Structure IC₅₀ (μM) 1 (curcumin)

8.2 ± 0.4 2

6.7 ± 1.2 3

3.4 ± 0.2 4

4.4 ± 0.8 5

5.0 ± 0.3Summary of Pertinent Initial Results

In summary, our initial studies (Weber et al., 2005, Bioorg Med Chem13:3811-3820; Weber et al., 2006, Biorg Med Chem 14:2450-2461) of thesynthesis and biological activities of curcumin analogs have providedlibraries of compounds, some of which are potent anti-oxidants, evenmore potent than the parent compounds, and some of which are devoid ofanti-oxidant activity. Our screening of curcumin analogs for theirabilities to prevent the TNF-α-induced activation of NF-κB in acell-based screening assay indicate that anti-oxidant activity is notnecessary for this biological activity, which suggests that there arespecific targets for these analogs. We are now in a position to utilizethese analogs in studies of EC dysfunction and to determine whetherinhibition of the activation of NF-κB is a promising approach todevelopment of new therapeutics for prevention of microvascularcomplications of diabetes.

Although activation of NF-κB is commonly observed in EC that are exposedto conditions associated with microvascular complications, and althoughthese treatments commonly result in up-regulation of a number ofpro-inflammatory genes as well as genes that promote leukocyte adhesion,endothelial permeability and matrix deposition, it remains to bedemonstrated that activation of NF-κB is essential for up-regulation ofthese genes. Therefore, we will examine the role for NF-κB in promotingthe alterations in EC gene expression associated with complications.

Our first goal is to demonstrate that activation of NF-κB precedes theup-regulation of genes associated with microvascular complications. Wewill utilize real-time PCR to monitor the expression of pro-inflammatoryfactors (IL-1, IL-6), extracellular matrix components (collagen,fibronectin), and leukocyte adhesion factors (E-selectin, ICAM-1). Theactivation of NF-κB will be analyzed by Western blots of nuclearextracts using p50/RelA-specific and p52/RelB-specific antibodies tomonitor both the classical and the alternative pathways. EC cells willbe exposed to high glucose, AGE, TNF-α, or angiotensin II to elicitestress. The AGE will include glucose-modified and methylglyoxal-modifiedalbumin prepared by both short- and long-term exposure of albumin tothese aldehydes. It is known that methylglyoxal is the main reactivealdehyde produced in diabetes and is the precursor to many of the knownAGE (Thornalley, 2002, Int Rev Neurobiol 50:37-57; Vander Jagt et al.,2003, Chem Biol Interact 143-144:341-351). In addition, AGE that formintracellularly in EC exposed to high glucose are derived frommethylglyoxal (Shinohara et al., 1998, J Clin Invest 101:1142-1147).Thus both AGE that are administered extracellularly and AGE that areproduced intracellularly may contribute to EC dysfunction. In addition,AGE prepared by short-term exposure of proteins to glucose and otheraldehydes elicit different effects than AGE prepared from long-termexposure (Mandl-Weber et al., 2001, Perit Dial Int 21:487-494;Schalkwijk et al., 2002, Semin Vasc Med 2:191-197). The experimentalcellular system will utilize human retinal EC isolated from humanretinas (NDR¹, Philadelphia). As an alternative, human umbilical veinendothelial cells (HUVEC), which are commercially available (Clonetics),will be compared to retinal EC. If the results of initial experimentsare similar, HUVEC will be used in place of retinal EC for convenience.

Our second goal is to determine whether activation of NF-κB is essentialfor up-regulation of this battery of genes. We will utilize siRNA tosilence selected components of the IKK/IκB/NF-κB system. As before,real-time PCR will be used to monitor expression of thepro-inflammatory, extracellular matrix and cell adhesion genes inresponse to the various stresses administered, except that the EC willfirst be treated with the appropriate siRNA. Real-time PCR and Westernanalysis will be used to demonstrate that the desired target has beensilenced. siRNA targets will include IKKβ and p50/RelA to monitor theclassical pathway and IKKα and p52/RelB for the alternative pathway.

Our third goal is to determine whether ROS must be increased in ECbefore there is activation of NF-κB. This question is related to theobservation that increases in ROS are commonly observed in studies ofthe activation of NF-κB. The production of ROS in response to exposureof EC to the various agents will be monitored by FACS analysis withROS-sensitive dyes such as dihydrorhodamine 123. Thus, the main questionis whether there is a required temporal relationship between exposure ofEC to stress, increase of ROS, activation of NF-κB, and up-regulation ofcomplications-associated genes.

We are developing libraries of compounds related to biologically active,polyphenolic natural products including curcumin. Thus, expansion of theanalog libraries already described (Weber et al., 2005, Bioorg Med Chem13:3811-3820; Weber et al., 2006, Biorg Med Chem 14:2450-2461) is anongoing effort that will provide a range of compounds for use in thisproject. Analogs of curcumin that are more active than the parentcompounds as inhibitors of the activation of NF-κB are available,including compounds devoid of anti-oxidant activity as well as analogsthat are stronger anti-oxidants than the parent compounds. This range ofanalogs will be used in studies of the stress response of EC. Inaddition, analogs are being developed that exhibit improvedbioavailability compared to curcumin, which shows poor bioavailability(Garcea et al., 2004, Br J Cancer 9:1011-1015). The poor bioavailabilityof curcumin is the main reason that attempts to develop this naturalproduct as a drug likely will not succeed. Therefore, there is a need todevelop analogs with better pharmacokinetic properties.

We will determine whether the active analogs identified with thePanomics NF-κB Reporter Stable Cell assay also prevent the activation ofNF-κB in EC in response to high glucose, AGE, angiotensin II and TNF-α.As before, we will follow the activation of NF-κB by Western analysis ofnuclear extracts using p50/RelA-specific or p52/RelB-specific antibodiesto assess the two pathways.

We will also determine whether inhibiting the activation of NF-κB in ECby analogs of curcumin prevents the up-regulation of genes associatedwith microvasculer complications. As before, we will utilize real-timePCR to monitor the expression of pro-inflammatory factors, extracellularmatrix components, and leukocyte adhesion factors. To quantitate theeffects of analogs on target expression, we will use the comparativeC_(T) method. The amount of target message with analog treatment will benormalized to the internal reference (β-actin) and compared to thecalibrator (target expression without analog treatment). Since curcuminis an established inhibitor of NF-κB, it will serve as our positivecontrols in all experiments. The 96-well plate format will permit a highefficiency and rapid screen to accurately assess individual analogs aswell as obtain quantitative data to determine individual K_(i) values.The Applied Biosystems 7000 System is capable of multiplexing 96 samplessimultaneously in approximately 2 h. Comparisons between cells treatedwith varying concentrations of analogs will be made by Student's t-test.Differences in p-values <0.05 will be considered significant.

We will also identify the site(s) of action of curcumin analogs inpreventing the activation of NF-κB in EC. A likely target is IKKβ orIKKα, based upon literature reports that implicate these targets (Bhartiet al., 2003, Blood 101:1053-1062). However, it is also possible that akinase upstream of IKK may be a target. We will initially screen analogsagainst recombinant human IKK (α and β) to test whether there is acorrelation between the cellular data and enzyme inhibition data foreither of these kinases. If neither IKK isoform is inhibited by theseanalogs, then additional kinases will need to be evaluated as likelytargets.

Materials and Methods

Cell culture and chemicals: EC will be isolated from retina dissectedfrom human eyes obtained from NDR1 (Philadelphia). Fresh retinas areincubated in DMEM with 0.01% type 1 collagenase, filtered through nylonmesh and the endothelial cells are isolated with antibody-coatedmagnetic beads for endothelial cells (DynabeadsCD31, Dynal Biotech) (Suet al., 2003, Molec Vision 9:171-178). EC are maintained in Dulbecco'smodified Eagle's medium (DMEM, low glucose formulation) supplementedwith 4 mM L-glutamine, 10% fetal bovine serum (FBS), 100 units/mlpenicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B). AGE(methylglyoxal (MeG)-modified HSA and glucose-modified HSA, both short(one week)- and long (seven week)-term glycation), are prepared understerile conditions. MeG is obtained as the dimethylacetal (Aldrich)because commercial MeG is contaminated with formaldehyde. MeG isliberated from the acetal under acid conditions and then purified byazeotropic distillation with water and standardized with theglyoxalase-I reaction, as we described (Vander Jagt et al., 2003, ChemBiol Interact 143-144:341-351).

Real-time PCR: EC will be plated in 96-well plates for assay (5×10⁴cells/well). When cells reach 80-90% confluency, they will be stressedwith 25 mM glucose, AGE, TNF-α or angiotensin II, together with varyingconcentrations of either curcumin or its analogs, for varying timesdepnding on the stressor, at 37° C. 5% CO₂/95% air. Total RNA will thenbe extracted, isolated using an RNeasy kit (Invitrogen) and quantitatedby measuring absorbance at 260 nm. One-step reverse transcriptase (RT)coupled to real time PCR analysis will be performed using an AppliedBiosystems 7000 System. Primers (designed to amplify <150 bp) and TaqManprobe for the various transcripst will be designed using AppliedBiosystems Primer Express software. Primers and TaqMan Probe for β-actinwill be used as an internal control. Real-time PCR values obtained forβ-actin will be used to normalize values for target expression tocorrect for loading or cell number differences between wells. Cyclingparameters will be determined to optimize target and β-actinamplifications. Our starting parameters have been successful foramplification of many different genes currently under study: 50° C. 10min (RT reaction), 94° C. 2 min (RT enzyme inactivation, Taq Polymeraseactivation), 40 cycles 92° C. 30 s, 60° C. 30 s, 72° C. 30 s.

Western analysis: Activation of NF-κB by the classical pathway will bedetermined with the Panomics TransBinding NF-κB Assay Kit, whichquantifies the activation of p50. This ELISA-based assay, which utilizesimmobilized oligonucleotide containing NF-κB consensus sites, can beused with whole cell extracts or nuclear extracts; nuclear extracts willbe prepared using the Panomics Nuclear Extraction Kit. Activation ofNF-κB by the alternative pathway will be determined by Western analysiswith human RelB antibody (abI2013, Ancam) as the primary antibody(rabbit polyclonal) and HRP-conjugated goat anti-rabbit IgG as thesecond antibody.

siRNA

Cellular oxidative stress: ROS determinations by FACS analysis will becarried out with dihydrorhodamine 123 (or alternatively with2′,7′-dichlorodihydrofluorescein). Cells from the various experimentalprotocols are treated with the nonfluorescent, permeable form of theindicator which is trapped intracellularly following uptake andhydrolysis. After oxidation of the dye to the fluorescent form byintracellular ROS, cells are analyzed by Becton Dickenson FACSCAN withexcitation 495, emission 525 nm, respectively, carried out in theUniversity New Mexico Flow Cytometry Facility. Cells are treated withstress-inducer for varying periods, incubated with dye for 30 min,trypsinized and then subjected to FACS, with at least 20,000 eventsanalyzed.

Synthesis: Our recent report on the anti-oxidant properties of curcuminand its analogs (Weber et al., 2005, Bioorg Med Chem 13:3811-3820)included description of a wide range of synthetic procedures asdescribed in FIG. 37. Currently there are about 100 analogs in ourcurcumin library.

Screening with the Panomics NF-κB Reporter Stable Cell: The followingprocedure was used to obtain the data reported recently (Weber et al.,2006, Biorg Med Chem 14:2450-2461). An NF-κB reporter stable cell linefrom human 293T embryonic kidney cells (293T/NFκB-luc) (Panomics) wasgrown in a humidified atmosphere at 37° C. in 5% CO₂/95% air. The cellswere maintained in Dulbecco's Modified Eagle's Medium (DMEM—high glucosecontaining 4 mM glutamine) supplemented with 10% fetal bovine serum(FBS), 1 mM sodium pyruvate, 100 units/ml penicillin, 100 μg/mlstreptomycin and 100 μg/ml hygromycin (Gibco/Invitrogen, Carlsbad,Calif.)) to maintain cell selection. One day prior to treatment, the293T/NFκB-luc cells were plated into 24-well cell culture plates(Costar) at approximately 70% confluency in the above media withouthygromycin. The following day cells were fed fresh media 1 hour prior totreatment. Media with or without recombinant tumor necrosis factor alpha(TNF-α) (R&D Biosciences/Clontech) was then applied to the cells at 20ng/ml followed by immediate treatment with curcumin or analog. The cellswere placed again in a humidified atmosphere at 37° C. in 5% CO₂/95% airfor 7 hours. Plate wells were gently washed with phosphate bufferedsaline, pH 7.4, and lysed with 1× passive lysis buffer (Promega). Thesubsequent lysates were analyzed with the Luciferase Assay System(Promega) utilizing a TD-20/20 luminometer (Turner Designs). The fireflyluciferase relative light units were normalized to protein (mg/ml) withBCA™ Protein Assay Kit (Pierce) and standardized to TNF-α control.

Screening against IKKα and IKKβ: It is likely that some of the analogsin the curcumin libraries may inhibit IKK, based upon reports in theliterature (Shimizu et al., 2005, Mutat Res 591:147-160; Bharti et al.,2003, Blood 101:1053-1062). Recombinant human IKKα and IKKβ are nowavailable (Upstate). Therefore, we will screen our library against thesetwo kinases. We anticipate that we may identify a subset of compoundsthat are inhibitors of these kinases. If this is the case, then thesesubsets of analogs will be used separately to develop IKK-specific SARto identify new, more potent inhibitors that may be highly specific foreach IKK. The IKK screening will be carried out as follows: Enzymereactions are conducted in 40 mM MOPS buffer, pH7, containing ImM EDTA.The substrate is a commercial IKK peptide substrate (named IKKtide) fromUpstate, used at 100 μM. The reaction is initiated with 100 μM[γ-³²P]ATP (3000 Ci/mmol, Perkin Elmer). Reactions mixtures with andwithout added analog are incubated for 10 min at 30° C., transferred toP81 paper, washed with 0.75% phosphoric acid, then acetone, and countedin standard scintillation cocktail. For analogs that are active, kineticruns to obtain dissociation constants will be conducted using a range ofIKKtide concentrations, with and without added analog. CPM will beconverted into initial rates and analyzed by non-linear regression(ENZFITTER, Elsevier-Biosoft).

Bioavailability: Analogs of curcumin that are promising drug candidatesfrom studies of inhibition of activation of NF-κB in EC cells will beexamined for predicted oral bioavailability by use of the ParallelArtificial Membrane Permeability Assay (PAMPA), which uses ahexadecane-filled membrane (Millipore MultiScreen Permeability Plate) asa lipophilic barrier in a 96-well format. This is then combined with a96-well plate reader for quantitation of material that passes throughthe membrane (Kansy et al., 1998, J Med Chem 41:1007-1010).

Anticipated Problems and Alternative Procedures: The syntheticstrategies are straightforward and have been used in our preliminarystudies; these will allow us to synthesize a wide variety of analogs.The determinations of ROS levels use standard procedures. HUVECS areavailable as a backup to EC. The various treatments (high glucose, AGE,TNF-α, angiotensin II) provide overlap between the two pathways foractivation of NF-κB and, therefore, some redundancy. The siRNA procedureis not anticipated to present any problems; use of antisenseoligonucleotides is available as a backup. Likewise the real-time PCRprocedure is not anticipated to present any problems; conventional useof Northerns is available as a backup.

Example 12 Glutathione-S-Transferase P1-1: An Anticancer Drug Target

Introduction

Glutathione-S-Transferase (GSTP1-1) belongs to theglutathione-S-transferase family of detoxification enzymes. It catalyzesthe addition of glutathione, an important biological antioxidant, totoxic electrophiles including anti-cancer drugs. GSTP1-1 has been shownto be over expressed in many cancers, which leads to increasedresistance to anti-cancer drugs. We propose that inhibition of GSTP1-1will increase susceptibility of the cancer to the anti-cancer drug.

Curcumin has been shown to inhibit GSTP1-1. Curcumin and analogues orcurcumin were analyzed for their ability to inhibit GSTP1-1 activity.

Materials and Methods

Computer modeling was used to screen 63 curcumin compounds for promisinginhibitors of GSTP1-1. Computer modeling used a crystal structure ofGSTP1-1 from the Protein Data Bank. Inhibitors were drawn using Sybylmolecular modeling software. Inhibitors were docked to GSTP1-1 usingAutodock3, which estimates the binding constant of the inhibitor to theprotein.

Kinetic analysis assays were conducted. In photometric assays, curcuminand 63 curcumin analogues were screened. GSTP1-1 activity was measuredat pH 6.5 in 1 ml volumes of 20 mM potassium phosphate buffer containing100 mM NaCl, 1 mM CDNB and 1 mM GSH. Curcumin and curcumin analogueswere screened at a concentration of 25 μM and the reaction was initiatedwith 70 ng of GSTP1-1 enzyme and monitored at 340 nm, 25° C.(Perkin/Elmer Lambda 2S spectrophotometer). These activities wereplotted as percent of control (GSTP1-1 activity without inhibitors).Kinetic analysis (dissociation constants) of inhibitors was determinedby linear regression analysis of the Dixon, plots. Routinely, assayswere carried out as above, however, at two different constantconcentrations of GSH (0.25 and 1.25 mM) varying the concentration ofanalogue. Ki's were calculated utilizing SigmaPlot's Enzyme KineticsModule™ (Chicago, Ill., USA).

Results

FIG. 38 and FIG. 39 show curcumin analogues #1 and #2, respectively, andsome estimated Ki binding constants from computer modeling with theirmolecular structures. Dixon plots for curcumin analogues #1 (left) and#2 (right) showing inhibition of GSTP1-1 are shown in FIG. 40. These twoanalogues were shown to exhibit better inhibition than curcumin. Kivalues for these analogues were similar to the predicted Ki values fromcomputer modeling.

Conclusions

In kinetic analysis experiments, assays of the natural product curcuminanalogues of curcumin were tested for inhibition of GSTP1-1. Experimentsshowed that curcumin analogues inhibit GSTP1-1 well. Many analogues ofcurcumin inhibit GSTP1-1 activity better than curcumin itself. Ingeneral, computer modeling predicted inhibition results that were inagreement with experimental results. Curcumin analogues that inhibitGSTP1-1 will be tested for their abilities to sensitize breast cancercells to anti-cancer drugs in culture.

Example 13 Inhibitory Activity of Curcumin Derivatives in GSTP1-1 Assayand Computer Modeling

Curcumin is known to inhibit GSTP1-1 activity. Therefore, it was thoughtthat modification of the structure of curcumin could lead to enhancedactivity. The library consisting of three series of curcumin analoguesexamined the role of the enone functionality in aryl systems where thespacer is 7-carbons (as in curcumin), 5-carbons or 3-carbons in length.In addition, the importance of aryl ring substituents was assessed. TheGSTP1-1 inhibitory activities of curcumin and analogues were determinedby the inhibition of the formation of product of a reaction between theelectrophilic 1-chloro-2,4-dinitrobenzene (CDNB) and the nucleophilicglutathione (GSH) at 340 nm which is catalyzed by GSTP1-1.

Curcumin and its analogues were tested for glutathione S-transferaseinhibitory activity by an enzyme assay. Glutathione S-transferaseactivity was measured at pH 6.5 in 1 ml volume aliquots of potassiumphosphate buffer (20 mM, Fluka; Sigma/Aldrich), containing sodiumchloride (100 mM, Fluka; Sigma/Aldrich), 1-chloro-2,4-dinitrobenzene(CDNB, 1 mM, Fluka; Sigma/Aldrich) and glutathione (GSH, 1 mM, Fluka;Sigma/Aldrich) followed by the addition of curcumin and analogues (10μl). The reaction was initiated with recombinant glutathioneS-transferase P1-1 enzyme (70 ng/ml final concentration, Calciochem) andmonitored at 340 nm at 25° C. for 1 min. These activities were plottedas percent of control (GSTP1-1 activity without inhibitors).

FIG. 41, FIG. 42 and FIG. 43 show analogues active in the GSTP1-1screening assay. The active analogues are arranged from highly active onthe left to slightly active on the right.

Active analogues in series 1, which contain a 7-carbon spacer, are shownin FIG. 41. Five analogues, 6b, 3b, 12b, 3c and 9b in this series weremore active than curcumin. Four of the active analogues in this seriescontain aryl groups with no substituents. Three of these analogues alsocontain a central methylene substituent with one of these analogues,12b, containing two benzyl substituents. The active analogues in thisseries are all very hydrophobic. There is very little correlation toantioxidant activity in these analogues.

Active analogues in series 2, which contain a 5-carbon spacer, are shownin FIG. 42. Twenty analogues including 29, 20aa, 20z, 31, 20ab, 20f,20y, 20x, 20p and 20b show better inhibitory activity than curcumin.Eight of the ten most active analogues contain an aryl group with asubstituent in either the para or meta position. Just as in the 7-carbonseries when the most active analogues were hydrophobic, analogues in the5-carbon series also follow this trend. Some of the active analogues inthis series also

Analogues in series 3, which contain a 3-carbon spacer, are shown inFIG. 43. No analogue in this series was more active than curcumin. Manyof the analogues in this series exhibit antioxidant activity.

We performed a similar assay using 2.5 mM glutathione and lmM1-chloro-2,4-dinitrobenzene as the electrophile. In this assay GSTP1-1was activated with TNFα. The reaction was monitored at 340 nm. Curcuminand each analogue were analyzed at 25 μM concentration. In that assay,it appeared that that analogues of curcumin in which the two aryl groupsare separated by 7-carbon, 5-carbon, or 3-carbon enone spacers are ableto inhibit the TNFα-induced activation of GSTP1-1 and, possibly,ultimately NFκB. However, activities can vary widely. The most activeanalogues are those that retain the enone functionality, so,surprisingly, it is found that this enone functionality is veryimportant and preferred for the inventive small molecules and its methodof application. Many of the analogues are more active than curcumin.Ring substituents are not necessary for activity but can affect it. Theanalogues need not have the same substituents on each of the aromatic orheterocyclic rings.

Computer Modeling

An additional docking study was performed on glutathione S-transferaseP1-1 (GSTP1-1). Curcumin is a known inhibitor of GSTP1-1. One crystalstructure was selected from the protein data bank of the thirty oneavailable selections. 19GS¹² was selected because it contained a largeportion of the GSTP1-1 protein and was complexed to its naturalsubstrate glutathione.

When glutathione was removed and docked back to GSTP1-1, glutathionebound in the same location and with the same orientation it had beforebeing removed from the protein. When the analogues were docked, they alldocked to the same binding area and most of the analogues docked intothe actual binding pocket. Many of these analogues have good K_(est)values with analogue 14a having the most potent K_(est) value at 3.51E-9 M as shown in Table 14. Nine of the docked analogues display betterK_(est) values than glutathione. Therefore, there is the potential thatany of these analogues could block glutathione from entering GSTP1-1.There is no correlation to the K_(exp) values.

TABLE 14 K_(est) Values for GSTP1-1 (19GS) 14a 3.51E−09  6a 1.09E−07 20i3.16E−07 40b 8.56E−07 15a 6.57E−09 52e 1.13E−07 20k 3.18E−07 46ak8.66E−07 15b 9.61E−09  3g 1.17E−07  3e 3.22E−07 45b 9.34E−07 12b1.26E−08 20l 1.20E−07 16b 3.37E−07 50b 9.37E−07  9b 3.33E−08  3b1.23E−07 20y 3.74E−07 20z 1.00E−06  9a 3.33E−08 20o 1.25E−07 20q3.92E−07 52ac 1.08E−06 17b 3.35E−08  3a 1.36E−07 36a 4.10E−07 311.09E−06 23 4.63E−08 13a 1.53E−07 20c 4.27E−07 52l 1.14E−06 53 4.79E−08 3d 1.64E−07 46a 4.41E−07 20p 1.24E−06 gluta 5.26E−08 20ab 1.77E−07 40af4.55E−07 20aa 1.29E−06 20ag 5.34E−08 20m 2.03E−07 20n 4.68E−07 20b1.33E−06 20w 5.67E−08 13b 2.19E−07  6b 4.72E−07 36e 1.40E−06 20v5.87E−08 45a 2.22E−07 20a 4.86E−07 29 1.45E−06 20ae 6.81E−08 20u2.25E−07 20r 5.30E−07 42b 1.57E−06 38a 6.84E−08 48ad 2.28E−07 20ac5.39E−07 20t 1.97E−06 14b 7.09E−08 20e 2.54E−07 48a 6.36E−07 39b2.06E−06  3i 7.75E−08 13c 2.61E−07 46ad 6.38E−07 34 2.57E−06 20g8.88E−08 52aa 2.67E−07 20f 7.40E−07 43b 3.25E−06 25 9.45E−08 11b2.68E−07 38b 7.61E−07 35a 4.00E−06  3f 9.68E−08 46al 2.80E−07 20s7.90E−07 35q 5.83E−06 20ah 1.05E−07 52b 2.84E−07 20x 8.01E−07 35e1.12E−05  3h 1.07E−07 20d 3.01E−07

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. Any inconsistency betweenthe material incorporated by reference and the material set for in thespecification as originally filed shall be resolved in favor of thespecification as originally filed. The foregoing detailed descriptionand examples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A method of treating a subject afflicted withAlzheimer's disease, the method comprising administering to the subjecta therapeutically effective amount of a composition comprising acurcumin derivative according to the chemical structure:


2. The method according to claim 1 wherein said compound is


3. The method according to claim 1 wherein said compound is


4. The method according to claim 1 wherein said compound is


5. The method of claim 1, wherein the composition further comprises apharmaceutically acceptable carrier.
 6. The method of claim 1, whereinthe curcumin derivative inhibits at least one of AP-1 or NF-κB.
 7. Themethod of claim 1, wherein the curcumin derivative inhibits amyloidplaque formation.
 8. The method of claim 1, wherein the curcuminderivative inhibits aggregation of a plurality of Aβ peptides.
 9. Themethod of claim 1, wherein the curcumin derivative inhibitsoligomerization of a plurality of Aβ peptides.
 10. The method of claim1, wherein the curcumin derivative decreases the cytotoxicity of an Aβpeptide aggregate.
 11. The method of claim 1, wherein the curcuminderivative decreases activation of a glial cell by an Aβ peptideaggregate.
 12. The method according to claim 1 wherein said curcuminderivative is a compound according to the chemical structure:


13. The method according to claim 1 wherein said curcumin derivative isa compound according to the chemical structure: